Chemically modified multifunctional short interfering nucleic acid molecules that mediate RNA interference

ABSTRACT

The present invention relates to a multifunctional short interfering nucleic acid (siNA) having a structure of Formula MF-III: 
     
       
         
         
             
             
         
       
     
     wherein each X, X′, Y, and Y′ is independently an oligonucleotide of length about 15 nucleotides to about 50 nucleotides; X comprises a nucleotide sequence that is complementary to a nucleotide sequence present in region Y′; X′ comprises a nucleotide sequence that is complementary to a nucleotide sequence present in region Y; one or more of X, X′, Y, and Y′ is independently complementary to a first, second, third, or fourth target sequence, respectively, or a portion thereof; and W represents a nucleotide or non-nucleotide linker that connects sequences Y′ and Y, wherein the siNA directs cleavage of the first, second, third, and/or fourth target sequence via RNA interference.

This application is a continuation of U.S. patent application Ser. No.14/712,733, filed May 14, 2015, which is a continuation of U.S. patentapplication Ser. No. 12/064,014, filed Aug. 17, 2006, which is a 371national stage entry of International Patent Application No.PCT/US06/032168, filed Aug. 17, 2006, which is a continuation-in-part ofU.S. patent application Ser. No. 11/299,254, filed Dec. 8, 2005, whichis a continuation-in-part of U.S. patent application Ser. No.11/234,730, filed Sep. 23, 2005, which is a continuation-in-part of U.S.patent application Ser. No. 11/205,646, filed Aug. 17, 2005, which is acontinuation-in-part of U.S. patent application Ser. No. 11/098,303,filed Apr. 4, 2005, which is a continuation-in-part of U.S. patentapplication Ser. No. 10/923,536, filed Aug. 20, 2004, which is acontinuation-in-part of International Patent Application No.PCT/US04/16390, filed May 24, 2004. The instant application claims thebenefit of all the listed applications, which are hereby incorporated byreference herein in their entireties, including the drawings.

FIELD OF THE INVENTION

The present invention relates to compounds, compositions, and methodsfor the study, diagnosis, and treatment of traits, diseases andconditions that respond to the modulation of gene expression and/oractivity. The present invention is also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in gene expression pathways or other cellular processes thatmediate the maintenance or development of such traits, diseases andconditions. Specifically, the invention relates to double strandednucleic acid molecules including small nucleic acid molecules, such asshort interfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and shmt hairpin RNA(shRNA) molecules capable of mediating RNA interference (RNAi) againstgene expression, including cocktails of such small nucleic acidmolecules and lipid nanoparticle (LNP) formulations of such smallnucleic acid molecules. The present invention also relates to smallnucleic acid molecules, such as siNA, siRNA, and others that can inhibitthe function of endogenous RNA molecules, such as endogenous micro-RNA(miRNA) (e.g, miRNA inhibitors) or endogenous short interfering RNA(siRNA), (e.g., siRNA inhibitors) or that can inhibit the function ofRISC (e.g., RISC inhibitors), to modulate gene expression by interferingwith the regulatory function of such endogenous RNAs or proteinsassociated with such endogenous RNAs (e.g., RISC), including cocktailsof such small nucleic acid molecules and lipid nanoparticle (LNP)formulations of such small nucleic acid molecules. Such small nucleicacid molecules and are useful, for example, in providing compositions toprevent, inhibit, or reduce various diseases, traits and conditions thatare associated with gene expression or activity in a subject ororganism.

BACKGROUND OF THE INVENTION

The following is a discussion of relevant art pertaining to RNAi. Thediscussion is provided only for understanding of the invention thatfollows. The summary is not an admission that any of the work describedbelow is prior art to the claimed invention.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Zamore et al., 2000, Cell, 101, 25-33; Fireet al., 1998, Nature, 391, 806; Hamilton et al., 1999, Science, 286,950-951; Lin et al., 1999, Nature, 402, 128-129; Sharp, 1999, genes &Dev., 13:139-141; and Strauss, 1999, Science, 286, 886). Thecorresponding process in plants (Heifetz et al., International PCTPublication No. WO 99/61631) is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire et al., 1999, Trendsgenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA or viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized. This mechanism appearsto be different from other known mechanisms involving double strandedRNA-specific ribonucleases, such as the interferon response that resultsfrom dsRNA-mediated activation of protein kinase PKR and2′,5′-oligoadenylate synthetase resulting in non-specific cleavage ofmRNA by ribonuclease L (see for example U.S. Pat. Nos. 6,107,094;5,898,031; Clemens et al., 1997, J. Interferon & Cytokine Res., 17,503-524; Adah et al., 2001, Curr. Med. Chem., 8, 1189).

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer (Bass, 2000, Cell, 101,235; Zamore et al., 2000, Cell, 101, 25-33; Hammond et al., 2000,Nature, 404, 293). Dicer is involved in the processing of the dsRNA intoshort pieces of dsRNA known as short interfering RNAs (siRNAs) (Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2000, Cell, 101, 235; Berstein etal., 2001, Nature, 409, 363). Short interfering RNAs derived from diceractivity are typically about 21 to about 23 nucleotides in length andcomprise about 19 base pair duplexes (Zamore et al., 2000, Cell, 101,25-33; Elbashir et al., 2001, genes Dev., 15, 188). Dicer has also beenimplicated in the excision of 21- and 22-nucleotide small temporal RNAs(stRNAs) from precursor RNA of conserved structure that are implicatedin translational control (Hutvagner et al., 2001, Science, 293, 834).The RNAi response also features an endonuclease complex, commonlyreferred to as an RNA-induced silencing complex (RISC), which mediatescleavage of single-stranded RNA having sequence complementary to theantisense strand of the siRNA duplex. Cleavage of the target RNA takesplace in the middle of the region complementary to the antisense strandof the siRNA duplex (Elbashir et al., 2001, genes Dev., 15, 188).

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans.Bahramian and Zarbl, 1999, Molecular and Cellular Biology, 19, 274-283and Wianny and Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAimediated by dsRNA in mammalian systems. Hammond et al., 2000, Nature,404, 293, describe RNAi in Drosophila cells transfected with dsRNA.Elbashir et al., 2001, Nature, 411, 494 and Tuschl et al., InternationalPCT Publication No. WO 01/75164, describe RNAi induced by introductionof duplexes of synthetic 21-nucleotide RNAs in cultured mammalian cellsincluding human embryonic kidney and HeLa cells. Recent work inDrosophila embryonic lysates (Elbashir et al., 2001, EMBO J, 20, 6877and Tuschl et al., International PCT Publication No. WO 01/75164) hasrevealed certain requirements for siRNA length, structure, chemicalcomposition, and sequence that are essential to mediate efficient RNAiactivity. These studies have shown that 21-nucleotide siRNA duplexes aremost active when containing 3′-terminal dinucleotide overhangs.Furthermore, complete substitution of one or both siRNA strands with2′-deoxy (2′-H) or 2′-O-methyl nucleotides abolishes RNAi activity,whereas substitution of the 3′-terminal siRNA overhang nucleotides with2′-deoxy nucleotides (2′-H) was shown to be tolerated. Single mismatchsequences in the center of the siRNA duplex were also shown to abolishRNAi activity. In addition, these studies also indicate that theposition of the cleavage site in the target RNA is defined by the 5′-endof the siRNA guide sequence rather than the 3′-end of the guide sequence(Elbashir et al., 2001, EMBO J., 20, 6877). Other studies have indicatedthat a 5′-phosphate on the target-complementary strand of a siRNA duplexis required for siRNA activity and that ATP is utilized to maintain the5′-phosphate moiety on the siRNA (Nykanen et al., 2001, Cell, 107, 309).

Studies have shown that replacing the 3′-terminal nucleotide overhangingsegments of a 21-mer siRNA duplex having two-nucleotide 3′-overhangswith deoxyribonucleotides does not have an adverse effect on RNAiactivity. Replacing up to four nucleotides on each end of the siRNA withdeoxyribonucleotides has been reported to be well tolerated, whereascomplete substitution with deoxyribonucleotides results in no RNAiactivity (Elbashir et al., 2001, EMBO J., 20, 6877 and Tuschl et al.,International PCT Publication No. WO 01/75164). In addition, Elbashir etal., supra, also report that substitution of siRNA with 2′-O-methylnucleotides completely abolishes RNAi activity. Li et al., InternationalPCT Publication No. WO 00/44914, and Beach et al., International PCTPublication No. WO 01/68836 preliminarily suggest that siRNA may includemodifications to either the phosphate-sugar backbone or the nucleosideto include at least one of a nitrogen or sulfur heteroatom, however,neither application postulates to what extent such modifications wouldbe tolerated in siRNA molecules, nor provides any further guidance orexamples of such modified siRNA. Kreutzer et al., Canadian PatentApplication No. 2,359,180, also describe certain chemical modificationsfor use in dsRNA constructs in order to counteract activation ofdouble-stranded RNA-dependent protein kinase PKR, specifically 2′-aminoor 2′-O-methyl nucleotides, and nucleotides containing a 2′-0 or 4′-Cmethylene bridge. However, Kreutzer et al. similarly fails to provideexamples or guidance as to what extent these modifications would betolerated in dsRNA molecules.

Parrish et al., 2000, Molecular Cell, 6, 1077-1087, tested certainchemical modifications targeting the unc-22 gene in C. elegans usinglong (>25 nt) siRNA transcripts. The authors describe the introductionof thiophosphate residues into these siRNA transcripts by incorporatingthiophosphate nucleotide analogs with T7 and T3 RNA polymerase andobserved that RNAs with two phosphorothioate modified bases also hadsubstantial decreases in effectiveness as RNAi. Further, Parrish et al.reported that phosphorothioate modification of more than two residuesgreatly destabilized the RNAs in vitro such that interference activitiescould not be assayed. Id. at 1081. The authors also tested certainmodifications at the 2′-position of the nucleotide sugar in the longsiRNA transcripts and found that substituting deoxynucleotides forribonucleotides produced a substantial decrease in interferenceactivity, especially in the case of Uridine to Thymidine and/or Cytidineto deoxy-Cytidine substitutions. Id. In addition, the authors testedcertain base modifications, including substituting, in sense andantisense strands of the siRNA, 4-thiouracil, 5-bromouracil,5-iodouracil, and 3-(aminoallyl)uracil for uracil, and inosine forguanosine. Whereas 4-thiouracil and 5-bromouracil substitution appearedto be tolerated, Parrish reported that inosine produced a substantialdecrease in interference activity when incorporated in either strand.Parrish also reported that incorporation of 5-iodouracil and3-(aminoallyl)uracil in the antisense strand resulted in a substantialdecrease in RNAi activity as well.

The use of longer dsRNA has been described. For example, Beach et al.,International PCT Publication No. WO 01/68836, describes specificmethods for attenuating gene expression using endogenously-deriveddsRNA. Tuschl et al., International PCT Publication No. WO 01/75164,describe a Drosophila in vitro RNAi system and the use of specific siRNAmolecules for certain functional genomic and certain therapeuticapplications; although Tuschl, 2001, Chem. Biochem., 2, 239-245, doubtsthat RNAi can be used to cure genetic diseases or viral infection due tothe danger of activating interferon response. Li et al., InternationalPCT Publication No. WO 00/44914, describe the use of specific long (141bp-488 bp) enzymatically synthesized or vector expressed dsRNAs forattenuating the expression of certain target genes. Zernicka-Goetz etal., International PCT Publication No. WO 01/36646, describe certainmethods for inhibiting the expression of particular genes in mammaliancells using certain long (550 bp-714 bp), enzymatically synthesized orvector expressed dsRNA molecules. Fire et al., International PCTPublication No. WO 99/32619, describe particular methods for introducingcertain long dsRNA molecules into cells for use in inhibiting geneexpression in nematodes. Plaetinck et al., International PCT PublicationNo. WO 00/01846, describe certain methods for identifying specific genesresponsible for conferring a particular phenotype in a cell usingspecific long dsRNA molecules. Mello et al., International PCTPublication No. WO 01/29058, describe the identification of specificgenes involved in dsRNA-mediated RNAi. Pachuck et al., International PCTPublication No. WO 00/63364, describe certain long (at least 200nucleotide) dsRNA constructs. Deschamps Depaillette et al.,International PCT Publication No. WO 99/07409, describe specificcompositions consisting of particular dsRNA molecules combined withcertain anti-viral agents. Waterhouse et al., International PCTPublication No. 99/53050 and 1998, PNAS, 95, 13959-13964, describecertain methods for decreasing the phenotypic expression of a nucleicacid in plant cells using certain dsRNAs. Driscoll et al., InternationalPCT Publication No. WO 01/49844, describe specific DNA expressionconstructs for use in facilitating gene silencing in targeted organisms.

Others have reported on various RNAi and gene-silencing systems. Forexample, Parrish et al., 2000, Molecular Cell, 6, 1077-1087, describespecific chemically-modified dsRNA constructs targeting the unc-22 geneof C. elegans. Grossniklaus, International PCT Publication No. WO01/38551, describes certain methods for regulating polycomb geneexpression in plants using certain dsRNAs. Churikov et al.,International PCT Publication No. WO 01/42443, describe certain methodsfor modifying genetic characteristics of an organism using certaindsRNAs. Cogoni et al, International PCT Publication No. WO 01/53475,describe certain methods for isolating a Neurospora silencing gene anduses thereof. Reed et al., International PCT Publication No. WO01/68836, describe certain methods for gene silencing in plants. Honeret al., International PCT Publication No. WO 01/70944, describe certainmethods of drug screening using transgenic nematodes as Parkinson'sDisease models using certain dsRNAs. Deak et al., International PCTPublication No. WO 01/72774, describe certain Drosophila-derived geneproducts that may be related to RNAi, in Drosophila. Arndt et al.,International PCT Publication No. WO 01/92513 describe certain methodsfor mediating gene suppression by using factors that enhance RNAi.Tuschl et al., International PCT Publication No. WO 02/44321, describecertain synthetic siRNA constructs. Pachuk et al., International PCTPublication No. WO 00/63364, and Satishchandran et al., InternationalPCT Publication No. WO 01/04313, describe certain methods andcompositions for inhibiting the function of certain polynucleotidesequences using certain long (over 250 bp), vector expressed dsRNAs.Echeverri et al., International PCT Publication No. WO 02/38805,describe certain C. elegans genes identified via RNAi. Kreutzer et al.,International PCT Publications Nos. WO 02/055692, WO 02/055693, and EP1144623 B1 describes certain methods for inhibiting gene expressionusing dsRNA. Graham et al., International PCT Publications Nos. WO99/49029 and WO 01/70949, and AU 4037501 describe certain vectorexpressed siRNA molecules. Fire et al., U.S. Pat. No. 6,506,559,describe certain methods for inhibiting gene expression in vitro usingcertain long dsRNA (299 bp-1033 bp) constructs that mediate RNAi.Martinez et al., 2002, Cell, 110, 563-574, describe certain singlestranded siRNA constructs, including certain 5′-phosphorylated singlestranded siRNAs that mediate RNA interference in Hela cells. Harborth etal., 2003, Antisense & Nucleic Acid Drug Development, 13, 83-105,describe certain chemically and structurally modified siRNA molecules.Chiu and Rana, 2003, RNA, 9, 1034-1048, describe certain chemically andstructurally modified siRNA molecules. Woolf et al., International PCTPublication Nos. WO 03/064626 and WO 03/064625 describe certainchemically modified dsRNA constructs. Hornung et al., 2005, NatureMedicine, 11, 263-270, describe the sequence-specific potent inductionof IFN-alpha by short interfering RNA in plasmacytoid dendritic cellsthrough TLR7. Judge et al., 2005, Nature Biotechnology, Publishedonline: 20 Mar. 2005, describe the sequence-dependent stimulation of themammalian innate immune response by synthetic siRNA. Yuki et al.,International PCT Publication Nos. WO 05/049821 and WO 04/048566,describe certain methods for designing short interfering RNA sequencesand certain short interfering RNA sequences with optimized activity.Saigo et al., US Patent Application Publication No. US20040539332,describe certain methods of designing oligO- or polynucleotidesequences, including short interfering RNA sequences, for achieving RNAinterference. Tei et al., International PCT Publication No. WO03/044188, describe certain methods for inhibiting expression of atarget gene, which comprises transfecting a cell, tissue, or individualorganism with a double-stranded polynucleotide comprising DNA and RNAhaving a substantially identical nucleotide sequence with at least apartial nucleotide sequence of the target gene.

Mattick, 2005, Science, 309, 1527-1528; Claverie, 2005, Science, 309,1529-1530; Sethupathy et al., 2006, RNA, 12, 192-197; and Czech, 2006NEJM, 354, 11: 1194-1195; Hutvagner et al., US 20050227256, and Tuschlet al., US 20050182005, all describe antisense molecules that caninhibit miRNA function via steric blocking and are all incorporated byreference herein in their entirety.

SUMMARY OF THE INVENTION

This invention relates to compounds, compositions, and methods usefulfor modulating the expression of genes, such as those genes associatedwith the development or maintenance of diseases, traits and conditionsthat are related to gene expression or activity, by RNA interference(RNAi), using short interfering nucleic acid (siNA) molecules. Thisinvention also relates to compounds, compositions, and methods usefulfor modulating the expression and activity of one or more genes involvedin pathways of gene expression and/or activity by RNA interference(RNAi) using small nucleic acid molecules. In particular, the instantinvention features small nucleic acid molecules, such as shortinterfering nucleic acid (siNA), short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), and short hairpin RNA(shRNA) molecules and methods used to modulate the expression of genesand/or other genes involved in pathways of gene expression and/oractivity.

The instant invention also relates to small nucleic acid molecules, suchas siNA, siRNA, and others that can inhibit the function of endogenousRNA molecules, such as endogenous micro-RNA (miRNA) (e.g, miRNAinhibitors) or endogenous short interfering RNA (siRNA), (e.g., siRNAinhibitors) or that can inhibit the function of RISC (e.g., RISCinhibitors), to modulate gene expression by interfering with theregulatory function of such endogenous RNAs or proteins associated withsuch endogenous RNAs (e.g., RISC). Such molecules are collectivelyreferred to herein as RNAi inhibitors.

A siNA or RNAi inhibitor of the invention can be unmodified orchemically-modified. A siNA or RNAi inhibitor of the instant inventioncan be chemically synthesized, expressed from a vector or enzymaticallysynthesized. The instant invention also features variouschemically-modified synthetic short interfering nucleic acid (siNA)molecules capable of modulating target gene expression or activity incells by RNA interference (RNAi). The instant invention also featuresvarious chemically-modified synthetic short nucleic acid (siNA)molecules capable of modulating RNAi activity in cells by interactingwith miRNA, siRNA, or RISC, and hence down regulating or inhibiting RNAinterference (RNAi), translational inhibition, or transcriptionalsilencing in a cell or organism. The use of chemically-modified siNAand/or RNAi inhibitors improves various properties of native siNAmolecules and/or RNAi inhibitors through increased resistance tonuclease degradation in vivo and/or through improved cellular uptake.Further, contrary to earlier published studies, siNA molecules of theinvention having multiple chemical modifications, including fullymodified siNA, retains its RNAi activity. Therefore, Applicant teachesherein chemically modified siRNA (generally referred to herein as siNA)that retains or improves upon the activity of native siRNA. The siNAmolecules of the instant invention provide useful reagents and methodsfor a variety of therapeutic, prophylactic, veterinary, diagnostic,target validation, genomic discovery, genetic engineering, andpharmacogenomic applications.

In one embodiment, the invention features one or more siNA moleculesand/or RNAi inhibitors and methods that independently or in combinationmodulate the expression of target genes encoding proteins, such asproteins that are associated with the maintenance and/or development ofdiseases, traits, disorders, and/or conditions as described herein orotherwise known in the art, such as genes encoding sequences comprisingthose sequences referred to by GenBank Accession Nos. shown in U.S.Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536,and PCT/US03/05028 all of which are incorporated by reference herein,referred to herein generally as “target” sequences. The descriptionbelow of the various aspects and embodiments of the invention isprovided with reference to exemplary target genes referred to herein asgene targets. The present invention is also directed to compounds,compositions, and methods relating to traits, diseases and conditionsthat respond to the modulation of expression and/or activity of genesinvolved in gene expression pathways or other cellular processes thatmediate the maintenance or development of such traits, diseases andconditions. However, such reference is meant to be exemplary only andthe various aspects and embodiments of the invention are also directedto other genes that express alternate target genes, such as mutanttarget genes, splice variants of target genes, target gene variants fromspecies to species or subject to subject, and other target pathway genesdescribed herein or otherwise known in the art. Such additional genescan be analyzed for target sites using the methods described herein forexemplary target genes and sequences herein. Thus, the modulation andthe effects of such modulation of the other genes can be performed asdescribed herein. In other words, the terms “target” and “target gene”as defined herein below and recited in the described embodiments, ismeant to encompass genes associated with the development and/ormaintenance of diseases, traits and conditions herein, such as geneswhich encode polypeptides, regulatory polynucleotides (e.g., miRNAs andsiRNAs), mutant genes, and splice variants of genes, as well as othergenes involved in pathways of gene expression and/or activity. Thus,each of the embodiments described herein with reference to the term“target” are applicable to all of the protein, peptide, polypeptide,and/or polynucleotide molecules covered by the term “target”, as thatterm is defined herein. Comprehensively, such gene targets are alsoreferred to herein generally as “target” sequences.

In one embodiment, the invention features a composition comprising twoor more different siNA molecules and/or RNAi inhibitors of the invention(e.g., siNA, duplex forming siNA, or multifunctional siNA or anycombination thereof) targeting different polynucleotide targets, such asdifferent regions of a target RNA or DNA (e.g., two different targetsites such as provided herein or any combination of targets or pathwaytargets) or both coding and non-coding targets. Such pools of siNAmolecules can provide increased therapeutic effect.

In one embodiment, the invention features a pool of two or moredifferent siNA molecules of the invention (e.g., siNA, duplex formingsiNA, or multifunctional siNA or any combination thereof) that havespecificity for different polynucleotide targets, such as differentregions of target RNA or DNA (e.g., two different target sites herein orany combination of targets or pathway targets) or both coding andnon-coding targets, wherein the pool comprises siNA molecules targetingabout 2, 3, 4, 5, 6, 7, 8, 9, 10 or more different targets.

Due to the potential for sequence variability of the genome acrossdifferent organisms or different subjects, selection of siNA moleculesfor broad therapeutic applications likely involve the conserved regionsof the gene. In one embodiment, the present invention relates to siNAmolecules and/or RNAi inhibitors that target conserved regions of thegenome or regions that are conserved across different targets. siNAmolecules and/or RNAi inhibitors designed to target conserved regions ofvarious targets enable efficient inhibition of target gene expression indiverse patient populations.

In one embodiment, the invention features a double stranded nucleic acidmolecule, such as an siNA molecule, where one of the strands comprisesnucleotide sequence having complementarity to a predetermined nucleotidesequence in a target nucleic acid molecule, or a portion thereof. Thepredetermined nucleotide sequence can be a nucleotide target sequence,such as a sequence described herein or known in the art. In anotherembodiment, the predetermined nucleotide sequence is a target sequenceor pathway target sequence as is known in the art.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, wherein saidsiNA molecule comprises about 15 to about 28 base pairs.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of atarget RNA, wherein said siNA molecule comprises about 15 to about 28base pairs.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage, of atarget RNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first strand and a second strand, each strand ofthe siNA molecule is about 18 to about 28 (e.g., about 18, 19, 20, 21,22, 23, 24, 25, 26, 27, or 28) nucleotides in length, the first strandof the siNA molecule comprises nucleotide sequence having sufficientcomplementarity to the target RNA for the siNA molecule to directcleavage of the target RNA via RNA interference, and the second strandof said siNA molecule comprises nucleotide sequence that iscomplementary to the first strand. In one specific embodiment, forexample, each strand of the siNA molecule is about 18 to about 27nucleotides in length.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that directs cleavage of atarget RNA via RNA interference (RNAi), wherein the double stranded siNAmolecule comprises a first strand and a second strand, each strand ofthe siNA molecule is about 18 to about 23 (e.g., about 18, 19, 20, 21,22, or 23) nucleotides in length, the first strand of the siNA moleculecomprises nucleotide sequence having sufficient complementarity to thetarget RNA for the siNA molecule to direct cleavage of the target RNAvia RNA interference, and the second strand of said siNA moleculecomprises nucleotide sequence that is complementary to the first strand.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a target RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 28 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the target RNA for thesiNA molecule to direct cleavage of the target RNA via RNA interference.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a target RNA via RNA interference (RNAi), whereineach strand of the siNA molecule is about 18 to about 23 nucleotides inlength; and one strand of the siNA molecule comprises nucleotidesequence having sufficient complementarity to the target RNA for thesiNA molecule to direct cleavage of the target RNA via RNA interference.

In one embodiment, the invention features a siNA molecule thatdown-regulates expression of a target gene or that directs cleavage of atarget RNA, for example, wherein the target gene or RNA comprisesprotein encoding sequence. In one embodiment, the invention features asiNA molecule that down-regulates expression of a target gene or thatdirects cleavage of a target RNA, for example, wherein the target geneor RNA comprises non-coding sequence or regulatory elements involved intarget gene expression (e.g., non-coding RNA, miRNA, stRNA etc.).

In one embodiment, a siNA of the invention is used to inhibit theexpression of target genes or a target gene family, wherein the genes orgene family sequences share sequence homology. Such homologous sequencescan be identified as is known in the art, for example using sequencealignments. siNA molecules can be designed to target such homologoussequences, for example using perfectly complementary sequences or byincorporating non-canonical base pairs, for example mismatches and/orwobble base pairs, that can provide additional target sequences. Ininstances where mismatches are identified, non-canonical base pairs (forexample, mismatches and/or wobble bases) can be used to generate siNAmolecules that target more than one gene sequence. In a non-limitingexample, non-canonical base pairs such as UU and CC base pairs are usedto generate siNA molecules that are capable of targeting sequences fordiffering polynucleotide targets that share sequence homology. As such,one advantage of using siNAs of the invention is that a single siNA canbe designed to include nucleic acid sequence that is complementary tothe nucleotide sequence that is conserved between the homologous genes.In this approach, a single siNA can be used to inhibit expression ofmore than one gene instead of using more than one siNA molecule totarget the different genes.

In one embodiment, the invention features a siNA molecule having RNAiactivity against target RNA (e.g., coding or non-coding RNA), whereinthe siNA molecule comprises a sequence complementary to any RNAsequence, such as those sequences having GenBank Accession Nos. shown inPCT/US03/05028, U.S. Provisional Patent Application No. 60/363,124,and/or U.S. Ser. No. 10/923,536, all of which are incorporated byreference herein. In another embodiment, the invention features a siNAmolecule having RNAi activity against target RNA, wherein the siNAmolecule comprises a sequence complementary to an RNA having variantencoding sequence, for example other mutant genes known in the art to beassociated with the maintenance and/or development of diseases, traits,disorders, and/or conditions described herein or otherwise known in theart. Chemical modifications as shown in Table I or otherwise describedherein can be applied to any siNA construct of the invention. In anotherembodiment, a siNA molecule of the invention includes a nucleotidesequence that can interact with nucleotide sequence of a target gene andthereby mediate silencing of target gene expression, for example,wherein the siNA mediates regulation of target gene expression bycellular processes that modulate the chromatin structure or methylationpatterns of the target gene and prevent transcription of the targetgene.

In one embodiment, siNA molecules of the invention are used to downregulate or inhibit the expression of proteins arising from haplotypepolymorphisms that are associated with a trait, disease or condition ina subject or organism. Analysis of genes, or protein or RNA levels canbe used to identify subjects with such polymorphisms or those subjectswho are at risk of developing traits, conditions, or diseases describedherein. These subjects are amenable to treatment, for example, treatmentwith siNA molecules of the invention and any other composition useful intreating diseases related to target gene expression. As such, analysisof protein or RNA levels can be used to determine treatment type and thecourse of therapy in treating a subject. Monitoring of protein or RNAlevels can be used to predict treatment outcome and to determine theefficacy of compounds and compositions that modulate the level and/oractivity of certain proteins associated with a trait, disorder,condition, or disease.

In one embodiment of the invention a siNA molecule comprises anantisense strand comprising a nucleotide sequence that is complementaryto a nucleotide sequence or a portion thereof encoding a target protein.The siNA further comprises a sense strand, wherein said sense strandcomprises a nucleotide sequence of a target gene or a portion thereof.

In another embodiment, a siNA molecule comprises an antisense regioncomprising a nucleotide sequence that is complementary to a nucleotidesequence encoding a target protein or a portion thereof. The siNAmolecule further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence of a target gene or a portion thereof.

In another embodiment, the invention features a siNA molecule comprisingnucleotide sequence, for example, nucleotide sequence in the antisenseregion of the siNA molecule that is complementary to a nucleotidesequence or portion of sequence of a target gene. In another embodiment,the invention features a siNA molecule comprising a region, for example,the antisense region of the siNA construct, complementary to a sequencecomprising a target gene sequence or a portion thereof.

In one embodiment, the sense region or sense strand of a siNA moleculeof the invention is complementary to that portion of the antisenseregion or antisense strand of the siNA molecule that is complementary toa target polynucleotide sequence.

In yet another embodiment, the invention features a siNA moleculecomprising a sequence, for example, the antisense sequence of the siNAconstruct, complementary to a sequence or portion of sequence comprisingsequence represented by GenBank Accession Nos. shown in PCT/US03/05028,U.S. Provisional Patent Application No. 60/363,124, and/or U.S. Ser. No.10/923,536, all of which are incorporated by reference herein. Chemicalmodifications in Table I and described herein can be applied to any siNAconstruct of the invention. LNP formulations described in Table IV canbe applied to any siNA molecule or combination of siNA molecules herein.

In one embodiment of the invention a siNA molecule comprises anantisense strand having about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein the antisense strand is complementary to a target RNA sequenceor a portion thereof, and wherein said siNA further comprises a sensestrand having about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and wherein saidsense strand and said antisense strand are distinct nucleotide sequenceswhere at least about 15 nucleotides in each strand are complementary tothe other strand.

In one embodiment, a siNA molecule of the invention (e.g., a doublestranded nucleic acid molecule) comprises an antisense (guide) strandhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary toa target RNA sequence or a portion thereof. In one embodiment, at least15 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides) of a target RNA sequence arecomplementary to the antisense (guide) strand of a siNA molecule of theinvention.

In one embodiment, a siNA molecule of the invention (e.g., a doublestranded nucleic acid molecule) comprises a sense (passenger) strandhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that comprise sequence ofa target RNA or a portion thereof. In one embodiment, at least 15nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) nucleotides of a target RNA sequence comprise thesense (passenger) strand of a siNA molecule of the invention.

In another embodiment of the invention a siNA molecule of the inventioncomprises an antisense region having about 15 to about 30 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides, wherein the antisense region is complementary to a targetDNA sequence, and wherein said siNA further comprises a sense regionhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, wherein said senseregion and said antisense region are comprised in a linear moleculewhere the sense region comprises at least about 15 nucleotides that arecomplementary to the antisense region.

In one embodiment, a siNA molecule of the invention has RNAi activitythat modulates expression of RNA encoded by a gene. Because genes canshare some degree of sequence homology with each other, siNA moleculescan be designed to target a class of genes by selecting sequences thatare either shared amongst different targets or alternatively that areunique for a specific target. Therefore, in one embodiment, the siNAmolecule can be designed to target conserved regions of targetpolynucleotide sequences having homology among several gene variants soas to target a class of genes with one siNA molecule. Accordingly, inone embodiment, the siNA molecule of the invention modulates theexpression of one or more target gene isoforms or variants in a subjector organism. In another embodiment, the siNA molecule can be designed totarget a sequence that is unique to a specific polynucleotide sequence(e.g., a single target gene isoform or single nucleotide polymorphism(SNP)) due to the high degree of specificity that the siNA moleculerequires to mediate RNAi activity.

In one embodiment, nucleic acid molecules of the invention that act asmediators of the RNA interference gene silencing response aredouble-stranded nucleic acid molecules. In another embodiment, the siNAmolecules of the invention consist of duplex nucleic acid moleculescontaining about 15 to about 30 base pairs between oligonucleotidescomprising about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. In yet anotherembodiment, siNA molecules of the invention comprise duplex nucleic acidmolecules with overhanging ends of about 1 to about 3 (e.g., about 1, 2,or 3) nucleotides, for example, about 21-nucleotide duplexes with about19 base pairs and 3′-terminal mononucleotide, dinucleotide, ortrinucleotide overhangs: In yet another embodiment, siNA molecules ofthe invention comprise duplex nucleic acid molecules with blunt ends,where both ends are blunt, or alternatively, where one of the ends isblunt.

In one embodiment, a double stranded nucleic acid (e.g., siNA) moleculecomprises nucleotide or non-nucleotide overhangs. By “overhang” is meanta terminal portion of the nucleotide sequence that is not base pairedbetween the two strands of a double stranded nucleic acid molecule (seefor example FIG. 6). In one embodiment, a double stranded nucleic acidmolecule of the invention can comprise nucleotide or non-nucleotideoverhangs at the 3′-end of one or both strands of the double strandednucleic acid molecule. For example, a double stranded nucleic acidmolecule of the invention can comprise a nucleotide or non-nucleotideoverhang at the 3′-end of the guide strand or antisense strand/region,the 3′-end of the passenger strand or sense strand/region, or both theguide strand or antisense strand/region and the passenger strand orsense strand/region of the double stranded nucleic acid molecule. Inanother embodiment, the nucleotide overhang portion of a double strandednucleic acid (siNA) molecule of the invention comprises 2′-O-methyl,2′-deoxy, 2′-deoxy-2′-fluoro, 2′-deoxy-2′-fluoroarabino (FANA), 4′-thio,2′-Q-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, universal base, acyclic, or 5-C-methylnucleotides. In another embodiment, the non-nucleotide overhang portionof a double stranded nucleic acid (siNA) molecule of the inventioncomprises glyceryl, abasic, or inverted deoxy abasic non-nucleotides.

In one embodiment, the nucleotides comprising the overhang portions of adouble stranded nucleic acid (e.g., siNA) molecule of the inventioncorrespond to the nucleotides comprising the target polynucleotidesequence of the siNA molecule. Accordingly, in such embodiments, thenucleotides comprising the overhang portion of a siNA molecule of theinvention comprise sequence based on the target polynucleotide sequencein which nucleotides comprising the overhang portion of the guide strandor antisense strand/region of a siNA molecule of the invention can becomplementary to nucleotides in the target polynucleotide sequence andnucleotides comprising the overhang portion of the passenger strand orsense strand/region of a siNA molecule of the invention can comprise thenucleotides in the target polynucleotide sequence. Such nucleotideoverhangs comprise sequence that would result from Dicer processing of anative dsRNA into siRNA.

In one embodiment, the nucleotides comprising the overhang portion of adouble stranded nucleic acid (e.g., siNA) molecule of the invention arecomplementary to the target polynucleotide sequence and are optionallychemically modified as described herein. As such, in one embodiment, thenucleotides comprising the overhang portion of the guide strand orantisense strand/region of a siNA molecule of the invention can becomplementary to nucleotides in the target polynucleotide sequence, i.e.those nucleotide positions in the target polynucleotide sequence thatare complementary to the nucleotide positions of the overhangnucleotides in the guide strand or antisense strand/region of a siNAmolecule. In another embodiment, the nucleotides comprising the overhangportion of the passenger strand or sense strand/region of a siNAmolecule of the invention can comprise the nucleotides in the targetpolynucleotide sequence, i.e. those nucleotide positions in the targetpolynucleotide sequence that correspond to same the nucleotide positionsof the overhang nucleotides in the passenger strand or sensestrand/region of a siNA molecule. In one embodiment, the overhangcomprises a two nucleotide (e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA;3′-CU; 3′-CG; 3′CC; 3′-UA; 3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG;3′-AC; 3′-TA; 3′-TU; 3′-TG; 3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhangthat is complementary to a portion of the target polynucleotidesequence. In one embodiment, the overhang comprises a two nucleotide(e.g., 3′-GA; 3′-GU; 3′-GG; 3′GC; 3′-CA; 3′-CU; 3′-CG; 3′CC; 3′-UA;3′-UU; 3′-UG; 3′UC; 3′-AA; 3′-AU; 3′-AG; 3′-AC; 3′-TA; 3′-TU; 3′-TG;3′-TC; 3′-AT; 3′-UT; 3′-GT; 3′-CT) overhang that is not complementary toa portion of the target polynucleotide sequence. In another embodiment,the overhang nucleotides of a siNA molecule of the invention are2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabino, and/or2′-deoxy-2′-fluoro nucleotides. In another embodiment, the overhangnucleotides of a siNA molecule of the invention are 2′-O-methylnucleotides in the event the overhang nucleotides are purine nucleotidesand/or 2′-deoxy-2′-fluoro nucleotides or 2′-deoxy-2′-fluoroarabinonucleotides in the event the overhang nucleotides are pyrimidinesnucleotides. In another embodiment, the purine nucleotide (when present)in an overhang of siNA molecule of the invention is 2′-O-methylnucleotides. In another embodiment, the pyrimidine nucleotide (whenpresent) in an overhang of siNA molecule of the invention are2′-deoxy-2′-fluoro or 2′-deoxy-2′-fluoroarabino nucleotides.

In one embodiment, the nucleotides comprising the overhang portion of adouble stranded nucleic acid (e.g., siNA) molecule of the invention arenot complementary to the target polynucleotide sequence and areoptionally chemically modified as described herein. In one embodiment,the overhang comprises a 3′-UU overhang that is not complementary to aportion of the target polynucleotide sequence. In another embodiment,the nucleotides comprising the overhanging portion of a siNA molecule ofthe invention are 2′-O-methyl nucleotides, 2′-deoxy-2′-fluoroarabinoand/or 2′-deoxy-2′-fluoro nucleotides.

In one embodiment, the double stranded nucleic molecule (e.g., siNA) ofthe invention comprises a two or three nucleotide overhang, wherein thenucleotides in the overhang are the same or different. In oneembodiment, the double stranded nucleic molecule (e.g., siNA) of theinvention comprises a two or three nucleotide overhang, wherein thenucleotides in the overhang are the same or different and wherein one ormore nucleotides in the overhang are chemically modified at the base,sugar and/or phosphate backbone.

In one embodiment, the invention features one or morechemically-modified siNA constructs having specificity for targetnucleic acid molecules, such as DNA, or RNA encoding a protein ornon-coding RNA associated with the expression of target genes. In oneembodiment, the invention features a RNA based siNA molecule (e.g., asiNA comprising 2′-OH nucleotides) having specificity for nucleic acidmolecules that includes one or more chemical modifications describedherein. Non-limiting examples of such chemical modifications includewithout limitation phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No.10/981,966 filed Nov. 5, 2004, incorporated by reference herein),“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, 2′-deoxy-2′-fluoroarabino (FANA, see for example Dowler etal., 2006, Nucleic Acids Research, 34, 1669-1675) and terminal glyceryland/or inverted deoxy abasic residue incorporation. These chemicalmodifications, when used in various siNA constructs, (e.g., RNA basedsiNA constructs), are shown to preserve RNAi activity in cells while atthe same time, dramatically increasing the serum stability of thesecompounds.

In one embodiment, a siNA molecule of the invention comprises chemicalmodifications described herein (e.g., 2′-O-methyl ribonucleotides,2′-deoxy-2′-fluoro ribonucleotides, 4′-thio ribonucleotides,2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxynucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, LNA) at theinternal positions of the siNA molecule. By “internal position” is meantthe base paired positions of a siNA duplex.

In one embodiment, a siNA molecule of the invention comprises modifiednucleotides while maintaining the ability to mediate RNAi. The modifiednucleotides can be used to improve in vitro or in vivo characteristicssuch as stability, activity, toxicity, immune response, and/orbioavailability. For example, a siNA molecule of the invention cancomprise modified nucleotides as a percentage of the total number ofnucleotides present in the siNA molecule. As such, a siNA molecule ofthe invention can generally comprise about 5% to about 100% modifiednucleotides (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modifiednucleotides). For example, in one embodiment, between about 5% to about100% (e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) ofthe nucleotide positions in a siNA molecule of the invention comprise anucleic acid sugar modification, such as a 2′-sugar modification, e.g.,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides,2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifiuoromethoxynucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides, or 2′-deoxynucleotides. In another embodiment, between about 5% to about 100%(e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modified nucleotides) of thenucleotide positions in a siNA molecule of the invention comprise anucleic acid base modification, such as inosine, purine, pyridin-4-one,pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxy benzene,3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines(e.g., 5-methylcytidine), 5-alkyluridines (e.g., ribothymidine),5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or6-alkylpyrimidines (e.g., 6-methyluridine), or propyne modifications. Inanother embodiment, between about 5% to about 100% (e.g., about 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100% modified nucleotides) of the nucleotide positionsin a siNA molecule of the invention comprise a nucleic acid backbonemodification, such as a backbone modification having Formula I herein.In another embodiment, between about 5% to about 100% (e.g., about 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or 100% modified nucleotides) of the nucleotidepositions in a siNA molecule of the invention comprise a nucleic acidsugar, base, or backbone modification or any combination thereof (e.g.,any combination of nucleic acid sugar, base, backbone or non-nucleotidemodifications herein). In one embodiment, a siNA molecule of theinvention comprises at least about 20%, 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% modifiednucleotides. The actual percentage of modified nucleotides present in agiven siNA molecule will depend on the total number of nucleotidespresent in the siNA. If the siNA molecule is single stranded, thepercent modification can be based upon the total number of nucleotidespresent in the single stranded siNA molecules. Likewise, if the siNAmolecule is double stranded, the percent modification can be based uponthe total number of nucleotides present in the sense strand, antisensestrand, or both the sense and antisense strands.

A siNA molecule of the invention can comprise modified nucleotides atvarious locations within the siNA molecule. In one embodiment, a doublestranded siNA molecule of the invention comprises modified nucleotidesat internal base paired positions within the siNA duplex. For example,internal positions can comprise positions from about 3 to about 19nucleotides from the 5′-end of either sense or antisense strand orregion of a 21 nucleotide siNA duplex having 19 base pairs and twonucleotide 3′-overhangs. In another embodiment, a double stranded siNAmolecule of the invention comprises modified nucleotides at non-basepaired or overhang regions of the siNA molecule. By “non-base paired” ismeant, the nucleotides are not base paired between the sense strand orsense region and the antisense strand or antisense region or the siNAmolecule. The overhang nucleotides can be complementary or base pairedto a corresponding target polynucleotide sequence (see for example FIG.6C). For example, overhang positions can comprise positions from about20 to about 21 nucleotides from the 5′-end of either sense or antisensestrand or region of a 21 nucleotide siNA duplex having 19 base pairs andtwo nucleotide 3′-overhangs. In another embodiment, a double strandedsiNA molecule of the invention comprises modified nucleotides atterminal positions of the siNA molecule. For example, such terminalregions include the 3′-position, 5′-position, for both 3′ and5′-positions of the sense and/or antisense strand or region of the siNAmolecule. In another embodiment, a double stranded siNA molecule of theinvention comprises modified nucleotides at base-paired or internalpositions, non-base paired or overhang regions, and/or terminal regions,or any combination thereof.

One aspect of the invention features a double-stranded short interferingnucleic acid (siNA) molecule that down-regulates expression of a targetgene or that directs cleavage of a target RNA. In one embodiment, thedouble stranded siNA molecule comprises one or more chemicalmodifications and each strand of the double-stranded siNA is about 21nucleotides long. In one embodiment, the double-stranded siNA moleculedoes not contain any ribonucleotides. In another embodiment, thedouble-stranded siNA molecule comprises one or more ribonucleotides. Inone embodiment, each strand of the double-stranded siNA moleculeindependently comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides,wherein each strand comprises about 15 to about 30 (e.g., about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotidesthat are complementary to the nucleotides of the other strand. In oneembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence or a portion thereof of the target gene, and the second strandof the double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence of the target gene or aportion thereof.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, comprising anantisense region, wherein the antisense region comprises a nucleotidesequence that is complementary to a nucleotide sequence of the targetgene or a portion thereof, and a sense region, wherein the sense regioncomprises a nucleotide sequence substantially similar to the nucleotidesequence of the target gene or a portion thereof. In one embodiment, theantisense region and the sense region independently comprise about 15 toabout 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) nucleotides, wherein the antisense region comprisesabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) nucleotides that are complementary tonucleotides of the sense region.

In another embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the target gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region.

In one embodiment, a siNA molecule of the invention comprises bluntends, i.e., ends that do not include any overhanging nucleotides. Forexample, a siNA molecule comprising modifications described herein(e.g., comprising nucleotides having Formulae I-VII or siNA constructscomprising “Stab 00”-“Stab 36” or “Stab 3F”-“Stab 36F” (Table I) or anycombination thereof) and/or any length described herein can compriseblunt ends or ends with no overhanging nucleotides.

In one embodiment, any siNA molecule of the invention can comprise oneor more blunt ends, i.e. where a blunt end does not have any overhangingnucleotides. In one embodiment, the blunt ended siNA molecule has anumber of base pairs equal to the number of nucleotides present in eachstrand of the siNA molecule. In another embodiment, the siNA moleculecomprises one blunt end, for example wherein the 5′-end of the antisensestrand and the 3′-end of the sense strand do not have any overhangingnucleotides. In another example, the siNA molecule comprises one bluntend, for example, wherein the 3′-end of the antisense strand and the5′-end of the sense strand do not have any overhanging nucleotides. Inanother example, a siNA molecule comprises two blunt ends, for example,wherein the 3′-end of the antisense strand and the 5′-end of the sensestrand as well as the 5′-end of the antisense strand and 3′-end of thesense strand do not have any overhanging nucleotides. A blunt ended siNAmolecule can comprise, for example, from about 15 to about 30nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 nucleotides). Other nucleotides present in a bluntended siNA molecule can comprise, for example, mismatches, bulges,loops, or wobble base pairs to modulate the activity of the siNAmolecule to mediate RNA interference.

By “blunt ends” is meant symmetric termini or termini of a doublestranded siNA molecule having no overhanging nucleotides. The twostrands of a double stranded siNA molecule align with each other withoutover-hanging nucleotides at the termini. For example, a blunt ended siNAconstruct comprises terminal nucleotides that are complementary betweenthe sense and antisense regions of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. The sense regioncan be connected to the antisense region via a linker molecule, such asa polynucleotide linker or a non-nucleotide linker.

In one embodiment, a double stranded nucleic acid molecule (e.g., siNA)molecule of the invention comprises ribonucleotides at positions thatmaintain or enhance RNAi activity. In one embodiment, ribonucleotidesare present in the sense strand or sense region of the siNA molecule,which can provide for RNAi activity by allowing cleavage of the sensestrand or sense region by an enzyme within the RISC (e.g.,ribonucleotides present at the position of passenger strand, sensestrand, or sense region cleavage, such as position 9 of the passengerstrand of a 19 base-pair duplex, which is cleaved in the RISC by AGO2enzyme, see, for example, Matranga et al., 2005, Cell, 123:1-114 andRand et al., 2005, Cell, 123:621-629). In another embodiment, one ormore (for example 1, 2, 3, 4 or 5) nucleotides at the 5′-end of theguide strand or guide region (also known as antisense strand orantisense region) of the siNA molecule are ribonucleotides.

In one embodiment, a double stranded nucleic acid molecule (e.g., siNA)molecule of the invention comprises one or more ribonucleotides atpositions within the passenger strand or passenger region (also known asthe sense strand or sense region) that allows cleavage of the passengerstrand or passenger region by an enzyme in the RISC complex, (e.g.,ribonucleotides present at the position of passenger strand, such asposition 9 of the passenger strand of a 19 base-pair duplex that iscleaved in the RISC, see, for example, Matranga et al., 2005, Cell,123:1-114 and Rand et al., 2005, Cell, 123:621-629).

In one embodiment, a siNA molecule of the invention contains at least 2,3, 4, 5, or more chemical modifications that can be the same ofdifferent. In one embodiment, a siNA molecule of the invention containsat least 2, 3, 4, 5, or more different chemical modifications.

In one embodiment, a siNA molecule of the invention is a double-strandedshort interfering nucleic acid (siNA), wherein the double strandednucleic acid molecule comprises about 15 to about 30 (e.g., about 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) basepairs, and wherein one or more (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) of the nucleotide positions in each strand of thesiNA molecule comprises a chemical modification. In another embodiment,the siNA contains at least 2, 3, 4, 5, or more different chemicalmodifications.

In one embodiment, the invention features double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, wherein thesiNA molecule comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) base pairs, andwherein each strand of the siNA molecule comprises one or more chemicalmodifications. In one embodiment, each strand of the double strandedsiNA molecule comprises at least two (e.g., 2, 3, 4, 5, or more)different chemical modifications, e.g., different nucleotide sugar,base, or backbone modifications. In another embodiment, one of thestrands of the double-stranded siNA molecule comprises a nucleotidesequence that is complementary to a nucleotide sequence of a target geneor a portion thereof, and the second strand of the double-stranded siNAmolecule comprises a nucleotide sequence substantially similar to thenucleotide sequence or a portion thereof of the target gene. In anotherembodiment, one of the strands of the double-stranded siNA moleculecomprises a nucleotide sequence that is complementary to a nucleotidesequence of a target gene or portion thereof, and the second strand ofthe double-stranded siNA molecule comprises a nucleotide sequencesubstantially similar to the nucleotide sequence or portion thereof ofthe target gene. In another embodiment, each strand of the siNA moleculecomprises about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides, and each strandcomprises at least about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides that arecomplementary to the nucleotides of the other strand. The target genecan comprise, for example, sequences referred to herein or incorporatedherein by reference. The gene can comprise, for example, sequencesreferred to by GenBank Accession number herein.

In one embodiment, each strand of a double stranded siNA molecule of theinvention comprises a different pattern of chemical modifications, suchas any “Stab 00”-“Stab 36” or “Stab 3F”-“Stab 36F” (Table I)modification patterns herein or any combination thereof. Non-limitingexamples of sense and antisense strands of such siNA molecules havingvarious modification patterns are shown in Table II and FIGS. 4 and 5.

In one embodiment, a siNA molecule of the invention comprises noribonucleotides. In another embodiment, a siNA molecule of the inventioncomprises one or more ribonucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more ribonucleotides).

In one embodiment, a siNA molecule of the invention comprises anantisense region comprising a nucleotide sequence that is complementaryto a nucleotide sequence of a target gene or a portion thereof, and thesiNA further comprises a sense region comprising a nucleotide sequencesubstantially similar to the nucleotide sequence of the target gene or aportion thereof. In another embodiment, the antisense region and thesense region each comprise about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides andthe antisense region comprises at least about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides that are complementary to nucleotides of the sense region.In one embodiment, each strand of the double stranded siNA moleculecomprises at least two (e.g., 2, 3, 4, 5, or more) different chemicalmodifications, e.g., different nucleotide sugar, base, or backbonemodifications. The target gene can comprise, for example, sequencesreferred to herein or incorporated by reference herein. In anotherembodiment, the siNA is a double stranded nucleic acid molecule, whereeach of the two strands of the siNA molecule independently compriseabout 15 to about 40 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 23, 33, 34, 35, 36, 37, 38, 39, or 40)nucleotides, and where one of the strands of the siNA molecule comprisesat least about 15 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or25 or more) nucleotides that are complementary to the nucleic acidsequence of the target gene or a portion thereof.

In one embodiment, a siNA molecule of the invention comprises a senseregion and an antisense region, wherein the antisense region comprises anucleotide sequence that is complementary to a nucleotide sequence ofRNA encoded by a target gene, or a portion thereof, and the sense regioncomprises a nucleotide sequence that is complementary to the antisenseregion. In one embodiment, the siNA molecule is assembled from twoseparate oligonucleotide fragments, wherein one fragment comprises thesense region and the second fragment comprises the antisense region ofthe siNA molecule. In another embodiment, the sense region is connectedto the antisense region via a linker molecule. In another embodiment,the sense region is connected to the antisense region via a linkermolecule, such as a nucleotide or non-nucleotide linker. In oneembodiment, each strand of the double stranded siNA molecule comprisesat least two (e.g., 2, 3, 4, 5, or more) different chemicalmodifications, e.g., different nucleotide sugar, base, or backbonemodifications. The target gene can comprise, for example, sequencesreferred herein or incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) 2′-deoxy-2′-fluoro pyrimidine modificatons (e.g.,where one or more or all pyrimidine (e.g., U or C) positions of the siNAare modified with 2′-deoxy-2′-fluoro nucleotides). In one embodiment,the 2′-deoxy-2′-fluoro pyrimidine modifications are present in the sensestrand. In one embodiment, the 2′-deoxy-2′-fluoro pyrimidinemodifications are present in the antisense strand. In one embodiment,the 2′-deoxy-2′-fluoro pyrimidine modifications are present in both thesense strand and the antisense strand of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) 2′-O-methyl purine modificatons (e.g., where one ormore or all purine (e.g., A or G) positions of the siNA are modifiedwith 2′-O-methyl nucleotides). In one embodiment, the 2′-O-methyl purinemodifications are present in the sense strand. In one embodiment, the2′-O-methyl purine modifications are present in the antisense strand. Inone embodiment, the 2′-O-methyl purine modifications are present in boththe sense strand and the antisense strand of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more) 2′-deoxy purine modificatons (e.g., where one ormore or all purine (e.g., A or G) positions of the siNA are modifiedwith 2′-deoxy nucleotides). In one embodiment, the 2′-deoxy purinemodifications are present in the sense strand. In one embodiment, the2′-deoxy purine modifications are present in the antisense strand. Inone embodiment, the 2′-deoxy purine modifications are present in boththe sense strand and the antisense strand of the siNA molecule.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the target gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the siNA molecule has one or moremodified pyrimidine and/or purine nucleotides. In one embodiment, eachstrand of the double stranded siNA molecule comprises at least two(e.g., 2, 3, 4, 5, or more) different chemical modifications, e.g.,different nucleotide sugar, base, or backbone modifications. In oneembodiment, the pyrimidine nucleotides in the sense region are2′-O-methyl pyrimidine nucleotides or 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In another embodiment, the pyrimidinenucleotides in the sense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In one embodiment, the pyrimidinenucleotides in the antisense region are 2′-deoxy-2′-fluoro pyrimidinenucleotides and the purine nucleotides present in the antisense regionare 2′-O-methyl or 2′-deoxy purine nucleotides. In another embodiment ofany of the above-described siNA molecules, any nucleotides present in anon-complementary region of the sense strand (e.g., overhang region) are2′-deoxy nucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule, and wherein thefragment comprising the sense region includes a terminal cap moiety atthe 5′-end, the 3′-end, or both of the 5′ and 3′ ends of the fragment.In one embodiment, the terminal cap moiety is an inverted deoxy abasicmoiety or glyceryl moiety. In one embodiment, each of the two fragmentsof the siNA molecule independently comprise about 15 to about 30 (e.g.,about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30)nucleotides. In another embodiment, each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides. In a non-limitingexample, each of the two fragments of the siNA molecule comprise about21 nucleotides.

In one embodiment, the invention features a siNA molecule comprising atleast one modified nucleotide, wherein the modified nucleotide is a2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-fluoroarabino,2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide,or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modifiednucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966,filed Nov. 5, 2004, incorporated by reference herein. In one embodiment,the invention features a siNA molecule comprising at least two (e.g., 2,3, 4, 5, 6, 7, 8, 9, 10, or more) modified nucleotides, wherein themodified nucleotide is selected from the group consisting of2′-deoxy-2′-fluoro nucleotide, 2′-deoxy-2′-fluoroarabino,2′-O-trifluoromethyl nucleotide, 2′-O-ethyl-trifluoromethoxy nucleotide,or 2′-O-difluoromethoxy-ethoxy nucleotide or any other modifiednucleoside/nucleotide described herein and in U.S. Ser. No. 10/981,966,filed Nov. 5, 2004, incorporated by reference herein. The modifiednucleotide/nucleoside can be the same or different. The siNA can be, forexample, about 15 to about 40 nucleotides in length. In one embodiment,all pyrimidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro,2′-deoxy-2′-fluoroarabino, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy, 4′-thiopyrimidine nucleotides. In one embodiment, the modified nucleotides inthe siNA include at least one 2′-deoxy-2′-fluoro cytidine or2′-deoxy-2′-fluoro uridine nucleotide. In another embodiment, themodified nucleotides in the siNA include at least one 2′-deoxy-2′-fluorocytidine and at least one 2′-deoxy-2′-fluoro uridine nucleotides. In oneembodiment, all uridine nucleotides present in the siNA are2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, all cytidinenucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified internucleotidic linkage, such as phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoronucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro pyrimidine nucleotides. In one embodiment,the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoro cytidine or 2′-deoxy-2′-fluoro uridine nucleotide. Inanother embodiment, the modified nucleotides in the siNA include atleast one 2′-fluoro cytidine and at least one 2′-deoxy-2′-fluoro uridinenucleotides. In one embodiment, all uridine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro uridine nucleotides. In one embodiment, allcytidine nucleotides present in the siNA are 2′-deoxy-2′-fluoro cytidinenucleotides. In one embodiment, all adenosine nucleotides present in thesiNA are 2′-deoxy-2′-fluoro adenosine nucleotides. In one embodiment,all guanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroguanosine nucleotides. The siNA can further comprise at least onemodified intemucleotidic linkage, such as a phosphorothioate linkage. Inone embodiment, the 2′-deoxy-2′-fluoronucleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a method of increasing thestability of a siNA molecule against cleavage by ribonucleasescomprising introducing at least one modified nucleotide into the siNAmolecule, wherein the modified nucleotide is a 2′-deoxy-2′-fluoroarabinonucleotide. In one embodiment, all pyrimidine nucleotides present in thesiNA are 2′-deoxy-2′-fluoroarabino pyrimidine nucleotides. In oneembodiment, the modified nucleotides in the siNA include at least one2′-deoxy-2′-fluoroarabino cytidine or 2′-deoxy-2′-fluoroarabino uridinenucleotide. In another embodiment, the modified nucleotides in the siNAinclude at least one 2′-fluoro cytidine and at least one2′-deoxy-2′-fluoroarabino uridine nucleotides. In one embodiment, alluridine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabinouridine nucleotides. In one embodiment, all cytidine nucleotides presentin the siNA are 2′-deoxy-2′-fluoroarabino cytidine nucleotides. In oneembodiment, all adenosine nucleotides present in the siNA are2′-deoxy-2′-fluoroarabino adenosine nucleotides. In one embodiment, allguanosine nucleotides present in the siNA are 2′-deoxy-2′-fluoroarabinoguanosine nucleotides. The siNA can further comprise at least onemodified intemucleotidic linkage, such as a phosphorothioate linkage. InOne embodiment, the 2′-deoxy-2′-fluoroarabinonueleotides are present atspecifically selected locations in the siNA that are sensitive tocleavage by ribonucleases, such as locations having pyrimidinenucleotides.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, comprising asense region and an antisense region, wherein the antisense regioncomprises a nucleotide sequence that is complementary to a nucleotidesequence of RNA encoded by the target gene or a portion thereof and thesense region comprises a nucleotide sequence that is complementary tothe antisense region, and wherein the purine nucleotides present in theantisense region comprise 2′-deoxy-purine nucleotides. In an alternativeembodiment, the purine nucleotides present in the antisense regioncomprise 2′-O-methyl purine nucleotides. In either of the aboveembodiments, the antisense region can comprise a phosphorothioateinternucleotide linkage at the 3′ end of the antisense region.Alternatively, in either of the above embodiments, the antisense regioncan comprise a glyceryl modification at the 3′ end of the antisenseregion. In another embodiment of any of the above-described siNAmolecules, any nucleotides present in a non-complementary region of theantisense strand (e.g., overhang region) are 2′-deoxy nucleotides.

in one embodiment, the antisense region of a siNA molecule of theinvention comprises sequence complementary to a portion of an endogenoustranscript having sequence unique to a particular disease or traitrelated allele in a subject or organism, such as sequence comprising asingle nucleotide polymorphism (SNP) associated with the disease ortrait specific allele. As such, the antisense region of a siNA moleculeof the invention can comprise sequence complementary to sequences thatare unique to a particular allele to provide specificity in mediatingselective RNAi against the disease, condition, or trait related allele.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that down-regulates expressionof a target gene or that directs cleavage of a target RNA, wherein thesiNA molecule is assembled from two separate oligonucleotide fragmentswherein one fragment comprises the sense region and the second fragmentcomprises the antisense region of the siNA molecule. In one embodiment,each strand of the double stranded siNA molecule is about 21 nucleotideslong where about 19 nucleotides of each fragment of the siNA moleculeare base-paired to the complementary nucleotides of the other fragmentof the siNA molecule, wherein at least two 3′ terminal nucleotides ofeach fragment of the siNA molecule are not base-paired to thenucleotides of the other fragment of the siNA molecule. In anotherembodiment, the siNA molecule is a double stranded nucleic acidmolecule, where each strand is about 19 nucleotide long and where thenucleotides of each fragment of the siNA molecule are base-paired to thecomplementary nucleotides of the other fragment of the siNA molecule toform at least about 15 (e.g., 15, 16, 17, 18, or 19) base pairs, whereinone or both ends of the siNA molecule are blunt ends. In one embodiment,each of the two 3′ terminal nucleotides of each fragment of the siNAmolecule is a 2′-deoxy-pyrimidine nucleotide, such as a2′-deoxy-thymidine. In one embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule is a 2′-O-methylpyrimidine nucleotide, such as a 2′-O-methyl uridine, cytidine, orthymidine. In another embodiment, all nucleotides of each fragment ofthe siNA molecule are base-paired to the complementary nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule of about 19 to about25 base pairs having a sense region and an antisense region, where about19 nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the target gene. Inanother embodiment, about 21 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the target gene. In any of the above embodiments, the 5′-endof the fragment comprising said antisense region can optionally includea phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits the expression ofa target RNA sequence, wherein the siNA molecule does not contain anyribonucleotides and wherein each strand of the double-stranded siNAmolecule is about 15 to about 30 nucleotides. In one embodiment, thesiNA molecule is 21 nucleotides in length. Examples ofnon-ribonucleotide containing siNA constructs are combinations ofstabilization chemistries shown in Table I in any combination ofSense/Antisense chemistries, such as Stab 7/8, Stab 7/11, Stab 8/8, Stab18/8, Stab 18/11, Stab 12/13, Stab 7/13, Stab 18/13, Stab 7/19, Stab8/19, Stab 18/19, Stab 7/20, Stab 8/20, Stab 18/20, Stab 7/32, Stab8/32, or Stab 18/32 (e.g., any siNA having Stab 7, 8, 11, 12, 13, 14,15, 17, 18, 19, 20, or 32 sense or antisense strands or any combinationthereof). Herein, numeric Stab chemistries can include both 2′-fluoroand 2′-OCF3 versions of the chemistries shown in Table I. For example,“Stab 7/8” refers to both Stab 7/8 and Stab 7F/8F etc. In oneembodiment, the invention features a chemically synthesized doublestranded RNA molecule that directs cleavage of a target RNA via RNAinterference, wherein each strand of said RNA molecule is about 15 toabout 30 nucleotides in length; one strand of the RNA molecule comprisesnucleotide sequence having sufficient complementarity to the target RNAfor the RNA molecule to direct cleavage of the target RNA via RNAinterference; and wherein at least one strand of the RNA moleculeoptionally comprises one or more chemically modified nucleotidesdescribed herein, such as without limitation deoxynucleotides,2′-O-methyl nucleotides, 2′-deoxy-2′-fluoro nucleotides,2′-deoxy-2′-fluoroarabino, 2′-O-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides, etc. or any combination thereof.

In one embodiment, a target RNA of the invention comprises sequenceencoding a protein.

In one embodiment, target RNA of the invention comprises non-coding RNAsequence (e.g., miRNA, snRNA, siRNA etc.), see for example Mattick,2005, Science, 309, 1527-1528; Claverie, 2005, Science, 309, 1529-1530;Sethupathy et al., 2006, RNA, 12, 192-197; and Czech, 2006 NEJM, 354,11: 1194-1195.

In one embodiment, the invention features a medicament comprising a siNAmolecule of the invention.

In one embodiment, the invention features an active ingredientcomprising a siNA molecule of the invention.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule to inhibit,down-regulate, or reduce expression of a target gene, wherein the siNAmolecule comprises one or more chemical modifications and each strand ofthe double-stranded siNA is independently about 15 to about 30 or more(e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29or 30 or more) nucleotides long. In one embodiment, the siNA molecule ofthe invention is a double stranded nucleic acid molecule comprising oneor more chemical modifications, where each of the two fragments of thesiNA molecule independently comprise about 15 to about 40 (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 23,33, 34, 35, 36, 37, 38, 39, or 40) nucleotides and where one of thestrands comprises at least 15 nucleotides that are complementary tonucleotide sequence of target encoding RNA or a portion thereof. In anon-limiting example, each of the two fragments of the siNA moleculecomprise about 21 nucleotides. In another embodiment, the siNA moleculeis a double stranded nucleic acid molecule comprising one or morechemical modifications, where each strand is about 21 nucleotide longand where about 19 nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule, wherein at least two 3′ terminal nucleotides of eachfragment of the siNA molecule are not base-paired to the nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule comprising one ormore chemical modifications, where each strand is about 19 nucleotidelong and where the nucleotides of each fragment of the siNA molecule arebase-paired to the complementary nucleotides of the other fragment ofthe siNA molecule to form at least about 15 (e.g., 15, 16, 17, 18, or19) base pairs, wherein one or both ends of the siNA molecule are bluntends. In one embodiment, each of the two 3′ terminal nucleotides of eachfragment of the siNA molecule is a 2′-deoxy-pyrimidine nucleotide, suchas a 2′-deoxy-thymidine. In one embodiment, each of the two 3′ terminalnucleotides of each fragment of the siNA molecule is a 2′-O-methylpyrimidine nucleotide, such as a 2′-O-methyl uridine, cytidine, orthymidine. In another embodiment, all nucleotides of each fragment ofthe siNA molecule are base-paired to the complementary nucleotides ofthe other fragment of the siNA molecule. In another embodiment, the siNAmolecule is a double stranded nucleic acid molecule of about 19 to about25 base pairs having a sense region and an antisense region andcomprising one or more chemical modifications, where about 19nucleotides of the antisense region are base-paired to the nucleotidesequence or a portion thereof of the RNA encoded by the target gene. Inanother embodiment, about 21 nucleotides of the antisense region arebase-paired to the nucleotide sequence or a portion thereof of the RNAencoded by the target gene. In any of the above embodiments, the 5′-endof the fragment comprising said antisense region can optionally includea phosphate group.

In one embodiment, the invention features the use of a double-strandedshort interfering nucleic acid (siNA) molecule that inhibits,down-regulates, or reduces expression of a target gene, wherein one ofthe strands of the double-stranded siNA molecule is an antisense strandwhich comprises nucleotide sequence that is complementary to nucleotidesequence of target RNA or a portion thereof, the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand. In one embodiment, eachstrand has at least two (e.g., 2, 3, 4, 5, or more) chemicalmodifications, which can be the same or different, such as nucleotide,sugar, base, or backbone modifications. In one embodiment, a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, a majority of thepurine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a target gene, wherein one of the strands ofthe double-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence oftarget RNA or a portion thereof, wherein the other strand is a sensestrand which comprises nucleotide sequence that is complementary to anucleotide sequence of the antisense strand. In one embodiment, eachstrand has at least two (e.g., 2, 3, 4, 5, or more) chemicalmodifications, which can be the same or different, such as nucleotide,sugar, base, or backbone modifications. In one embodiment, a majority ofthe pyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, a majority of thepurine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits, down-regulates,or reduces expression of a target gene, wherein one of the strands ofthe double-stranded siNA molecule is an antisense strand which comprisesnucleotide sequence that is complementary to nucleotide sequence oftarget RNA that encodes a protein or portion thereof, the other strandis a sense strand which comprises nucleotide sequence that iscomplementary to a nucleotide sequence of the antisense strand andwherein a majority of the pyrimidine nucleotides present in thedouble-stranded siNA molecule comprises a sugar modification. In oneembodiment, each strand of the siNA molecule comprises about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides, wherein each strand comprises atleast about 15 nucleotides that are complementary to the nucleotides ofthe other strand. In one embodiment, the siNA molecule is assembled fromtwo oligonucleotide fragments, wherein one fragment comprises thenucleotide sequence of the antisense strand of the siNA molecule and asecond fragment comprises nucleotide sequence of the sense region of thesiNA molecule. In one embodiment, the sense strand is connected to theantisense strand via a linker molecule, such as a polynucleotide linkeror a non-nucleotide linker. In a further embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-deoxy purine nucleotides. In another embodiment, the pyrimidinenucleotides present in the sense strand are 2′-deoxy-2′fluoro pyrimidinenucleotides and the purine nucleotides present in the sense region are2′-O-methyl purine nucleotides. In still another embodiment, thepyrimidine nucleotides present in the antisense strand are2′-deoxy-2′-fluoro pyrimidine nucleotides and any purine nucleotidespresent in the antisense strand are 2′-deoxy purine nucleotides. Inanother embodiment, the antisense strand comprises one or more2′-deoxy-2′-fluoro pyrimidine nucleotides and one or more 2′-O-methylpurine nucleotides. In another embodiment, the pyrimidine nucleotidespresent in the antisense strand are 2′-deoxy-2′-fluoro pyrimidinenucleotides and any purine nucleotides present in the antisense strandare 2′-O-methyl purine nucleotides. In a further embodiment the sensestrand comprises a 3′-end and a 5′-end, wherein a terminal cap moiety(e.g., an inverted deoxy abasic moiety or inverted deoxy nucleotidemoiety such as inverted thymidine) is present at the 5′-end, the 3′-end,or both of the 5′ and 3′ ends of the sense strand. In anotherembodiment, the antisense strand comprises a phosphorothioateinternucleotide linkage at the 3′ end of the antisense strand. Inanother embodiment, the antisense strand comprises a glycerylmodification at the 3′ end. In another embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In any of the above-described embodiments of a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of atarget gene, wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, eachof the two strands of the siNA molecule can comprise about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides. In one embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule. In another embodiment, about 15 toabout 30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 or more) nucleotides of each strand of thesiNA molecule are base-paired to the complementary nucleotides of theother strand of the siNA molecule, wherein at least two 3′ terminalnucleotides of each strand of the siNA molecule are not base-paired tothe nucleotides of the other strand of the siNA molecule. In anotherembodiment, each of the two 3′ terminal nucleotides of each fragment ofthe siNA molecule is a 2′-deoxy-pyrimidine, such as 2′-deoxy-thymidine.In one embodiment, each strand of the siNA molecule is base-paired tothe complementary nucleotides of the other strand of the siNA molecule.In one embodiment, about 15 to about 30 (e.g., about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides of theantisense strand are base-paired to the nucleotide sequence of thetarget RNA or a portion thereof. In one embodiment, about 18 to about 25(e.g., about 18, 19, 20, 21, 22, 23, 24, or 25) nucleotides of theantisense strand are base-paired to the nucleotide sequence of thetarget RNA or a portion thereof.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of atarget gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of target RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand. In one embodiment, each strand has at least two (e.g., 2, 3, 4,5, or more) different chemical modifications, such as nucleotide sugar,base, or backbone modifications. In one embodiment, a majority of thepyrimidine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, a majority of thepurine nucleotides present in the double-stranded siNA moleculecomprises a sugar modification. In one embodiment, the 5′-end of theantisense strand optionally includes a phosphate group.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of atarget gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of target RNA or a portionthereof, the other strand is a sense strand which comprises nucleotidesequence that is complementary to a nucleotide sequence of the antisensestrand and wherein a majority of the pyrimidine nucleotides present inthe double-stranded siNA molecule comprises a sugar modification, andwherein the nucleotide sequence or a portion thereof of the antisensestrand is complementary to a nucleotide sequence of the untranslatedregion or a portion thereof of the target RNA.

In one embodiment, the invention features a double-stranded shortinterfering nucleic acid (siNA) molecule that inhibits expression of atarget gene, wherein one of the strands of the double-stranded siNAmolecule is an antisense strand which comprises nucleotide sequence thatis complementary to nucleotide sequence of target RNA or a portionthereof, wherein the other strand is a sense strand which comprisesnucleotide sequence that is complementary to a nucleotide sequence ofthe antisense strand, wherein a majority of the pyrimidine nucleotidespresent in the double-stranded siNA molecule comprises a sugarmodification, and wherein the nucleotide sequence of the antisensestrand is complementary to a nucleotide sequence of the target RNA or aportion thereof that is present in the target RNA.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention and a pharmaceutically acceptable carrieror diluent. In another embodiment, the invention features two or morediffering siNA molecules of the invention (e.g., siNA molecules thattarget different regions of target RNA or siNA molecules that targetSREBP1 pathway RNA) and a pharmaceutically acceptable carrier ordiluent.

In a non-limiting example, the introduction of chemically-modifiednucleotides into nucleic acid molecules provides a powerful tool inovercoming potential limitations of in vivo stability andbioavailability inherent to native RNA molecules that are deliveredexogenously. For example, the use of chemically-modified nucleic acidmolecules can enable a lower dose of a particular nucleic acid moleculefor a given therapeutic effect since chemically-modified nucleic acidmolecules tend to have a longer half-life in serum. Furthermore, certainchemical modifications can improve the bioavailability of nucleic acidmolecules by targeting particular cells or tissues and/or improvingcellular uptake of the nucleic acid molecule. Therefore, even if theactivity of a chemically-modified nucleic acid molecule is reduced ascompared to a native nucleic acid molecule, for example, when comparedto an all-RNA nucleic acid molecule, the overall activity of themodified nucleic acid molecule can be greater than that of the nativemolecule due to improved stability and/or delivery of the molecule.Unlike native unmodified siNA, chemically-modified siNA can alsominimize the possibility of activating interferon activity orimmunostimulation in humans. These properties therefore improve uponnative siRNA or minimally modified siRNA's ability to mediate RNAi invarious in vitro and in vivo settings, including use in both researchand therapeutic applications. Applicant describes herein chemicallymodified siNA molecules with improved RNAi activity compared tocorresponding unmodified or minimally modified siRNA molecules. Thechemically modified siNA motifs disclosed herein provide the capacity tomaintain RNAi activity that is substantially similar to unmodified orminimally modified active siRNA (see for example Elbashir et al., 2001,EMBO J., 20:6877-6888) while at the same time providing nucleaseresistance and pharmacoketic properties suitable for use in therapeuticapplications.

In any of the embodiments of siNA molecules described herein, theantisense region of a siNA molecule of the invention can comprise aphosphorothioate internucleotide linkage at the 3′-end of said antisenseregion. In any of the embodiments of siNA molecules described herein,the antisense region can comprise about one to about fivephosphorothioate internucleotide linkages at the 5′-end of saidantisense region. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs of a siNA molecule of theinvention can comprise ribonucleotides or deoxyribonucleotides that arechemically-modified at a nucleic acid sugar, base, or backbone. In anyof the embodiments of siNA molecules described herein, the 3′-terminalnucleotide overhangs can comprise one or more universal baseribonucleotides. In any of the embodiments of siNA molecules describedherein, the 3′-terminal nucleotide overhangs can comprise one or moreacyclic nucleotides.

One embodiment of the invention provides an expression vector comprisinga nucleic acid sequence encoding at least one siNA molecule of theinvention in a manner that allows expression of the nucleic acidmolecule. Another embodiment of the invention provides a mammalian cellcomprising such an expression vector. The mammalian cell can be a humancell. The siNA molecule of the expression vector can comprise a senseregion and an antisense region. The antisense region can comprisesequence complementary to a RNA or DNA sequence encoding a target andthe sense region can comprise sequence complementary to the antisenseregion. The siNA molecule can comprise two distinct strands havingcomplementary sense and antisense regions. The siNA molecule cancomprise a single strand having complementary sense and antisenseregions.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides comprising a backbonemodified internucleotide linkage having Formula I:

wherein each R1 and R2 is independently any nucleotide, non-nucleotide,or polynucleotide which can be naturally-occurring orchemically-modified and which can be included in the structure of thesiNA molecule or serve as a point of attachment to the siNA molecule,each X and Y is independently O, S, N, alkyl, or substituted alkyl, eachZ and W is independently O, S, N, alkyl, substituted alkyl, O-alkyl,S-alkyl, alkaryl, aralkyl, or acetyl and wherein W, X, Y, and Z areoptionally not all O. In another embodiment, a backbone modification ofthe invention comprises a phosphonoacetate and/or thiophosphonoacetateinternucleotide linkage (see for example Sheehan et al., 2003, NucleicAcids Research, 31, 4109-4118).

The chemically-modified internucleotide linkages having Formula I, forexample, wherein any Z, W, X, and/or Y independently comprises a sulphuratom, can be present in one or both oligonucleotide strands of the siNAduplex, for example, in the sense strand, the antisense strand, or bothstrands. The siNA molecules of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemically-modifiedinternucleotide linkages having Formula I at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of the sense strand, the antisense strand, orboth strands. For example, an exemplary siNA molecule of the inventioncan comprise about 1 to about 5 or more (e.g., about 1, 2, 3, 4, 5, ormore) chemically-modified internucleotide linkages having Formula I atthe 5′-end of the sense strand, the antisense strand, or both strands.In another non-limiting example, an exemplary siNA molecule of theinvention can comprise one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, or more) pyrimidine nucleotides with chemically-modifiedinternucleotide linkages having Formula I in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purine nucleotideswith chemically-modified internucleotide linkages having Formula I inthe sense strand, the antisense strand, or both strands. In anotherembodiment, a siNA molecule of the invention having internucleotidelinkage(s) of Formula I also comprises a chemically-modified nucleotideor non-nucleotide having any of Formulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula II:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH,S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3,NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalkdylamino, substituted silyl, or a group having any of Formula I,II, III, IV, V, VI and/or VII, any of which can be included in thestructure of the siNA molecule or serve as a point of attachment to thesiNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidicbase such as adenine, guanine, uracil, cytosine, thymine,2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any othernon-naturally occurring base that can be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine. In oneembodiment, a nucleotide of the invention having Formula II is a2′-deoxy-2′-fluoro nucleotide. In one embodiment, a nucleotide of theinvention having Formula II is a 2′-O-methyl nucleotide. In oneembodiment, a nucleotide of the invention having Formula II is a2′-deoxy nucleotide.

The chemically-modified nucleotide or non-nucleotide of Formula II canbe present in one or both oligonucleotide strands of the siNA duplex,for example in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula II at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotides ornon-nucleotides of Formula II at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotides or non-nucleotides of Formula II at the 3′-end of the sensestrand, the antisense strand, or both strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) nucleotides or non-nucleotideshaving Formula III:

wherein each R3, R4, R5, R6, R7, R8, R10, R11 and R12 is independentlyH, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F, Cl, Br, CN, CF3,OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl, S-alkenyl,N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH, O-alkyl-SH,S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2, NO2, N3,NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl, O-aminoacid,O-aminoacyl, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,polyalklylamino, substituted silyl, or a group having any of Formula I,II, III, IV, V, VI and/or VII, any of which can be included in thestructure of the siNA molecule or serve as a point of attachment to thesiNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, and B is a nucleosidicbase such as adenine, guanine, uracil, cytosine, thymine,2-aminoadenosine, 5-methylcytosine, 2,6-diaminopurine, or any othernon-naturally occurring base that can be employed to be complementary ornon-complementary to target RNA or a non-nucleosidic base such asphenyl, naphthyl, 3-nitropyrrole, 5-nitroindole, nebularine, pyridone,pyridinone, or any other non-naturally occurring universal base that canbe complementary or non-complementary to target RNA. In one embodiment,R3 and/or R7 comprises a conjugate moiety and a linker (e.g., anucleotide or non-nucleotide linker as described herein or otherwiseknown in the art). Non-limiting examples of conjugate moieties includeligands for cellular receptors, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spermine or spermidine.

The chemically-modified nucleotide or non-nucleotide of Formula III canbe present in one or both oligonucleotide strands of the siNA duplex,for example, in the sense strand, the antisense strand, or both strands.The siNA molecules of the invention can comprise one or morechemically-modified nucleotides or non-nucleotides of Formula III at the3′-end, the 5′-end, or both of the 3′ and 5′-ends of the sense strand,the antisense strand, or both strands. For example, an exemplary siNAmolecule of the invention can comprise about 1 to about 5 or more (e.g.,about 1, 2, 3, 4, 5, or more) chemically-modified nucleotide(s) ornon-nucleotide(s) of Formula III at the 5′-end of the sense strand, theantisense strand, or both strands. In anther non-limiting example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) chemically-modifiednucleotide or non-nucleotide of Formula III at the 3′-end of the sensestrand, the antisense strand, or both strands.

In another embodiment, a siNA molecule of the invention comprises anucleotide having Formula II or III, wherein the nucleotide havingFormula II or III is in an inverted configuration. For example, thenucleotide having Formula II or III is connected to the siNA constructin a 3′-3′, 3′-2′, 2′-3′, or 5′-5′ configuration, such as at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro systemwherein the chemical modification comprises a 5′-terminal phosphategroup having Formula IV:

wherein each X and Y is independently O, S, N, alkyl, substituted alkyl,or alkylhalo; wherein each Z and W is independently O, S, N, alkyl,substituted alkyl, O-alkyl, S-alkyl, alkaryl, aralkyl, alkylhalo, oracetyl; and wherein W, X, Y and Z are optionally not all O and Y servesas a point of attachment to the siNA molecule.

In one embodiment, the invention features a siNA molecule having a5′-terminal phosphate group having Formula IV on thetarget-complementary strand, for example, a strand complementary to atarget RNA, wherein the siNA molecule comprises an all RNA siNAmolecule. In another embodiment, the invention features a siNA moleculehaving a 5′-terminal phosphate group having Formula IV on thetarget-complementary strand wherein the siNA molecule also comprisesabout 1 to about 3 (e.g., about 1, 2, or 3) nucleotide 3′-terminalnucleotide overhangs having about 1 to about 4 (e.g., about 1, 2, 3, or4) deoxyribonucleotides on the 3′-end of one or both strands. In anotherembodiment, a 5′-terminal phosphate group having Formula IV is presenton the target-complementary strand of a siNA molecule of the invention,for example a siNA molecule having chemical modifications having any ofFormulae I-VII.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises one or more phosphorothioateintemucleotide linkages. For example, in a non-limiting example, theinvention features a chemically-modified short interfering nucleic acid(siNA) having about 1, 2, 3, 4, 5, 6, 7, 8 or more phosphorothioateinternucleotide linkages in one siNA strand. In yet another embodiment,the invention features a chemically-modified short interfering nucleicacid (siNA) individually having about 1, 2, 3, 4, 5, 6, 7, 8 or morephosphorothioate internucleotide linkages in both siNA strands. Thephosphorothioate internucleotide linkages can be present in one or botholigonucleotide strands of the siNA duplex, for example in the sensestrand, the antisense strand, or both strands. The siNA molecules of theinvention can comprise one or more phosphorothioate internucleotidelinkages at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand, the antisense strand, or both strands. For example, anexemplary siNA molecule of the invention can comprise about 1 to about 5or more (e.g., about 1, 2, 3, 4, 5, or more) consecutivephosphorothioate internucleotide linkages at the 5′-end of the sensestrand, the antisense strand, or both strands. In another non-limitingexample, an exemplary siNA molecule of the invention can comprise one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) pyrimidinephosphorothioate intemucleotide linkages in the sense strand, theantisense strand, or both strands. In yet another non-limiting example,an exemplary siNA molecule of the invention can comprise one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) purinephosphorothioate internucleotide linkages in the sense strand, theantisense strand, or both strands.

Each strand of the double stranded siNA molecule can have one or morechemical modifications such that each strand comprises a differentpattern of chemical modifications. Several non-limiting examples ofmodification schemes that could give rise to different patterns ofmodifications are provided herein.

In one embodiment, the invention features a siNA molecule, wherein thesense strand comprises one or more, for example, about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more phosphorothioate internucleotide linkages, and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy and/or aboutone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifhioromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoroniethoxy-ethoxy, 4′-thioand/or 2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′- and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe sense strand comprises about 1 to about 5, specifically about 1, 2,3, 4, or 5 phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, or more) universal base modified nucleotides, and optionallya terminal cap molecule at the 3-end, the 5′-end, or both of the 3′- and5′-ends of the sense strand; and wherein the antisense strand comprisesabout 1 to about 5 or more, specifically about 1, 2, 3, 4, 5, or morephosphorothioate internucleotide linkages, and/or one or more (e.g.,about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy, 2′-O-methyl,2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more, pyrimidine nucleotides of the sense and/or antisense siNAstrand are chemically-modified with 2′-deoxy, 2′-O-methyl,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or 2′-deoxy-2′-fluoronucleotides, with or without about 1 to about 5 or more, for exampleabout 1, 2, 3, 4, 5, or more phosphorothioate internucleotide linkagesand/or a terminal cap molecule at the 3′-end, the 5′-end, or both of the3′- and 5′-ends, being present in the same or different strand.

In one embodiment, the invention features a siNA molecule, wherein theantisense strand comprises one or more, for example, about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, or more phosphorothioate internucleotide linkages,and/or about one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore) 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-0 ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe sense strand; and wherein the antisense strand comprises about 1 toabout 10 or more, specifically about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore phosphorothioate internucleotide linkages, and/or one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) 2′-deoxy,2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)universal base modified nucleotides, and optionally a terminal capmolecule at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends ofthe antisense strand. In another embodiment, one or more, for exampleabout 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine nucleotides ofthe sense and/or antisense siNA strand are chemically-modified with2′-deoxy, 2′-O-methyl, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without one or more, forexample, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more phosphorothioateinternucleotide linkages and/or a terminal cap molecule at the 3′-end,the 5′-end, or both of the 3′ and 5′-ends, being present in the same ordifferent strand.

In another embodiment, the invention features a siNA molecule, whereinthe antisense strand comprises about 1 to about 5 or more, specificallyabout 1, 2, 3, 4, 5 or more phosphorothioate internucleotide linkages,and/or one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more)2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/orone or more (e.g, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universalbase modified nucleotides, and optionally a terminal cap molecule at the3′-end, the 5′-end, or both of the 3′- and 5′-ends of the sense strand;and wherein the antisense strand comprises about 1 to about 5 or more,specifically about 1, 2, 3, 4, 5 or more phosphorothioateinternucleotide linkages, and/or one or more (e.g., about 1, 2, 3, 4, 5,6, 7, 8, 9, 10 or more 2′-deoxy, 2′-O-methyl, 2′-deoxy-2′-fluoro,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy, 4′-thio and/or one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more) universal base modified nucleotides,and optionally a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends of the antisense strand. In anotherembodiment, one or more, for example about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10or more pyrimidine nucleotides of the sense and/or antisense siNA strandare chemically-modified with 2′-deoxy, 2′-O-trifluoromethyl, 2′-O-ethyltrifluoromethoxy, 2′-O-difluoromethoxy-ethoxy, 4′-thio and/or2′-deoxy-2′-fluoro nucleotides, with or without about 1 to about 5, forexample about 1, 2, 3, 4, 5 or more phosphorothioate internucleotidelinkages and/or a terminal cap molecule at the 3′-end, the 5′-end, orboth of the 3′- and 5′-ends, being present in the same or differentstrand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule having about 1 to about 5 ormore (specifically about 1, 2, 3, 4, 5 or more) phosphorothioateinternucleotide linkages in each strand of the siNA molecule.

In another embodiment, the invention features a siNA molecule comprising2′-5′ internucleotide linkages. The 2′-5′ internucleotide linkage(s) canbe at the 3′-end, the 5′-end, or both of the 3′- and 5′-ends of one orboth siNA sequence strands. In addition, the 2′-5′ internucleotidelinkage(s) can be present at various other positions within one or bothsiNA sequence strands, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a pyrimidinenucleotide in one or both strands of the siNA molecule can comprise a2′-5′ internucleotide linkage, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,or more including every internucleotide linkage of a purine nucleotidein one or both strands of the siNA molecule can comprise a 2′-5′internucleotide linkage.

In another embodiment, a chemically-modified siNA molecule of theinvention comprises a duplex having two strands, one or both of whichcan be chemically-modified, wherein each strand is independently about15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, or 30) nucleotides in length, wherein the duplex hasabout 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the chemicalmodification comprises a structure having any of Formulae I-VII. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a duplex having two strands, one or both of which can bechemically-modified with a chemical modification having any of FormulaeI-VII or any combination thereof, wherein each strand consists of about21 nucleotides, each having a 2-nucleotide 3′-terminal nucleotideoverhang, and wherein the duplex has about 19 base pairs. In anotherembodiment, a siNA molecule of the invention comprises a single strandedhairpin structure, wherein the siNA is about 36 to about 70 (e.g., about36, 40, 45; 50, 55, 60, 65, or 70) nucleotides in length having about 15to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30) base pairs, and wherein the siNA can include achemical modification comprising a structure having any of FormulaeI-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a linearoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the linear oligonucleotide forms a hairpin structurehaving about 19 to about 21 (e.g., 19, 20, or 21) base pairs and a2-nucleotide 3′-terminal nucleotide overhang. In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.For example, a linear hairpin siNA molecule of the invention is designedsuch that degradation of the loop portion of the siNA molecule in vivocan generate a double-stranded siNA molecule with 3′-terminal overhangs,such as 3′-terminal nucleotide overhangs comprising about 2 nucleotides.

In another embodiment, a siNA molecule of the invention comprises ahairpin structure, wherein the siNA is about 25 to about 50 (e.g., about25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length having about 3to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, and wherein thesiNA can include one or more chemical modifications comprising astructure having any of Formulae I-VII or any combination thereof. Forexample, an exemplary chemically-modified siNA molecule of the inventioncomprises a linear oligonucleotide having about 25 to about 35 (e.g.,about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35) nucleotides that ischemically-modified with one or more chemical modifications having anyof Formulae I-VII or any combination thereof, wherein the linearoligonucleotide forms a hairpin structure having about 3 to about 25(e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 1.3, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, or 25) base pairs and a 5′-terminal phosphategroup that can be chemically modified as described herein (for example a5′-terminal phosphate group having Formula IV). In another embodiment, alinear hairpin siNA molecule of the invention contains a stem loopmotif, wherein the loop portion of the siNA molecule is biodegradable.In one embodiment, a linear hairpin siNA molecule of the inventioncomprises a loop portion comprising a non-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric hairpin structure, wherein the siNA is about 25 to about 50(e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in lengthhaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs, andwherein the siNA can include one or more chemical modificationscomprising a structure having any of Formulae I-VII or any combinationthereof. For example, an exemplary chemically-modified siNA molecule ofthe invention comprises a linear oligonucleotide having about 25 toabout 35 (e.g., about 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, or 35)nucleotides that is chemically-modified with one or more chemicalmodifications having any of Formulae I-VII or any combination thereof,wherein the linear oligonucleotide forms an asymmetric hairpin structurehaving about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25) base pairs and a5′-terminal phosphate group that can be chemically modified as describedherein (for example a 5′-terminal phosphate group having Formula IV). Inone embodiment, an asymmetric hairpin siNA molecule of the inventioncontains a stem loop motif, wherein the loop portion of the siNAmolecule is biodegradable. In another embodiment, an asymmetric hairpinsiNA molecule of the invention comprises a loop portion comprising anon-nucleotide linker.

In another embodiment, a siNA molecule of the invention comprises anasymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length, whereinthe sense region is about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides in length, wherein the sense region and the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. For example, anexemplary chemically-modified siNA molecule of the invention comprisesan asymmetric double stranded structure having separate polynucleotidestrands comprising sense and antisense regions, wherein the antisenseregion is about 18 to about 23 (e.g., about 18, 19, 20, 21, 22, or 23)nucleotides in length and wherein the sense region is about 3 to about15 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15)nucleotides in length, wherein the sense region the antisense regionhave at least 3 complementary nucleotides, and wherein the siNA caninclude one or more chemical modifications comprising a structure havingany of Formulae I-VII or any combination thereof. In another embodiment,the asymmetric double stranded siNA molecule can also have a 5′-terminalphosphate group that can be chemically modified as described herein (forexample a 5′-terminal phosphate group having Formula IV).

In another embodiment, a siNA molecule of the invention comprises acircular nucleic acid molecule, wherein the siNA is about 38 to about 70(e.g., about 38, 40, 45, 50, 55, 60, 65, or 70) nucleotides in lengthhaving about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30) base pairs, and wherein the siNA caninclude a chemical modification, which comprises a structure having anyof Formulae I-VII or any combination thereof. For example, an exemplarychemically-modified siNA molecule of the invention comprises a circularoligonucleotide having about 42 to about 50 (e.g., about 42, 43, 44, 45,46, 47, 48, 49, or 50) nucleotides that is chemically-modified with achemical modification having any of Formulae I-VII or any combinationthereof, wherein the circular oligonucleotide forms a dumbbell shapedstructure having about 19 base pairs and 2 loops.

In another embodiment, a circular siNA molecule of the inventioncontains two loop motifs, wherein one or both loop portions of the siNAmolecule is biodegradable. For example, a circular siNA molecule of theinvention is designed such that degradation of the loop portions of thesiNA molecule in vivo can generate a double-stranded siNA molecule with3′-terminal overhangs, such as 3′-terminal nucleotide overhangscomprising about 2 nucleotides.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) abasic moiety,for example a compound having Formula V:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingany of Formula I, II, III, IV, V, VI and/or VII, any of which can beincluded in the structure of the siNA molecule or serve as a point ofattachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2. Inone embodiment, R3 and/or R7 comprises a conjugate moiety and a linker(e.g., a nucleotide or non-nucleotide linker as described herein orotherwise known in the art). Non-limiting examples of conjugate moietiesinclude ligands for cellular receptors, such as peptides derived fromnaturally occurring protein ligands; protein localization sequences,including cellular ZIP code sequences; antibodies; nucleic acidaptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; steroids, and polyamines, such as PEI,spermine or spermidine.

In one embodiment, a siNA molecule of the invention comprises at leastone (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) inverted abasicmoiety, for example a compound having Formula VI:

wherein each R3, R4, R5, R6, R7, R8, R10, R11, R12, and R13 isindependently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl, F,Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl, O-alkenyl,S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH, O-alkyl-OH,O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl, alkyl-O-alkyl, ONO2,NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl, ONH2, O-aminoalkyl,O-aminoacid, O-aminoacyl, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalklylamino, substituted silyl, or a group havingany of Formula I, II, III, IV, V, VI and/or VII, any of which can beincluded in the structure of the siNA molecule or serve as a point ofattachment to the siNA molecule; R9 is O, S, CH2, S═O, CHF, or CF2, andeither R2, R3, R8 or R13 serve as points of attachment to the siNAmolecule of the invention. In one embodiment, R3 and/or R7 comprises aconjugate moiety and a linker (e.g., a nucleotide or non-nucleotidelinker as described herein or otherwise known in the art). Non-limitingexamples of conjugate moieties include ligands for cellular receptors,such as peptides derived from naturally occurring protein ligands;protein localization sequences, including cellular ZIP code sequences;antibodies; nucleic acid aptamers; vitamins and other co-factors, suchas folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

In another embodiment, a siNA molecule of the invention comprises atleast one (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)substituted polyalkyl moieties, for example a compound having FormulaVII:

wherein each n is independently an integer from 1 to 12, each R1, R2 andR3 is independently H, OH, alkyl, substituted alkyl, alkaryl or aralkyl,F, Cl, Br, CN, CF3, OCF3, OCH3, OCN, O-alkyl, S-alkyl, N-alkyl,O-alkenyl, S-alkenyl, N-alkenyl, SO-alkyl, alkyl-OSH, alkyl-OH,O-alkyl-OH, O-alkyl-SH, S-alkyl-OH, S-alkyl-SH, alkyl-S-alkyl,alkyl-O-alkyl, ONO2, NO2, N3, NH2, aminoalkyl, aminoacid, aminoacyl,ONH2, O-aminoalkyl, O-aminoacid, O-aminoacyl, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalklylamino, substituted silyl,or a group having any of Formula I, II, III, IV, V, VI and/or VII, anyof which can be included in the structure of the siNA molecule or serveas a point of attachment to the siNA molecule. In one embodiment, R3and/or R1 comprises a conjugate moiety and a linker (e.g., a nucleotideor non-nucleotide linker as described herein or otherwise known in theart). Non-limiting examples of conjugate moieties include ligands forcellular receptors, such as peptides derived from naturally occurringprotein ligands; protein localization sequences, including cellular ZIPcode sequences; antibodies; nucleic acid aptamers; vitamins and otherco-factors, such as folate and N-acetylgalactosamine; polymers, such aspolyethyleneglycol (PEG); phospholipids; cholesterol; steroids, andpolyamines, such as PEI, spermine or spermidine.

By “ZIP code” sequences is meant, any peptide or protein sequence thatis involved in cellular topogenic signaling mediated transport (see forexample Ray et al., 2004, Science, 306(1501): 1505).

Each nucleotide within the double stranded siNA molecule canindependently have a chemical modification comprising the structure ofany of Formulae I-VIII. Thus, in one embodiment, one or more nucleotidepositions of a siNA molecule of the invention comprises a chemicalmodification having structure of any of Formulae I-VII or any othermodification herein. In one embodiment, each nucleotide position of asiNA molecule of the invention comprises a chemical modification havingstructure of any of Formulae I-VII or any other modification herein.

In one embodiment, one or more nucleotide positions of one or bothstrands of a double stranded siNA molecule of the invention comprises achemical modification having structure of any of Formulae I-VII or anyother modification herein. In one embodiment, each nucleotide positionof one or both strands of a double stranded siNA molecule of theinvention comprises a chemical modification having structure of any ofFormulae I-VII or any other modification herein.

In another embodiment, the invention features a compound having FormulaVII, wherein R1 and R2 are hydroxyl (OH) groups, n=1, and R3 comprises 0and is the point of attachment to the 3′-end, the 5′-end, or both of the3′ and 5′-ends of one or both strands of a double-stranded siNA moleculeof the invention or to a single-stranded siNA molecule of the invention.This modification is referred to herein as “glyceryl” (for examplemodification 6 in FIG. 10).

In another embodiment, a chemically modified nucleoside ornon-nucleoside (e.g., a moiety having any of Formula V, VI or VII) ofthe invention is at the 3′-end, the 5′-end, or both of the 3′ and5′-ends of a siNA molecule of the invention. For example, chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) can be present at the 3′-end, the 5′-end, or both of the 3′and 5′-ends of the antisense strand, the sense strand, or both antisenseand sense strands of the siNA molecule. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the 5′-end and 3′-end of the sensestrand and the 3′-end of the antisense strand of a double stranded siNAmolecule of the invention. In one embodiment, the chemically modifiednucleoside or non-nucleoside (e.g., a moiety having Formula V, VI orVII) is present at the terminal position of the 5′-end and 3′-end of thesense strand and the 3′-end of the antisense strand of a double strandedsiNA molecule of the invention. In one embodiment, the chemicallymodified nucleoside or non-nucleoside (e.g., a moiety having Formula V,VI or VII) is present at the two terminal positions of the 5′-end and3′-end of the sense strand and the 3′-end of the antisense strand of adouble stranded siNA molecule of the invention. In one embodiment, thechemically modified nucleoside or non-nucleoside (e.g., a moiety havingFormula V, VI or VII) is present at the penultimate position of the5′-end and 3′-end of the sense strand and the 3′-end of the antisensestrand of a double stranded siNA molecule of the invention. In addition,a moiety having Formula VII can be present at the 3′-end or the 5′-endof a hairpin siNA molecule as described herein.

In another embodiment, a siNA molecule of the invention comprises anabasic residue having Formula V or VI, wherein the abasic residue havingFormula VI or VI is connected to the siNA construct in a 3′-3′, 3′-2′,2′-3′, or 5′-5′ configuration, such as at the 3′-end, the 5′-end, orboth of the 3′ and 5′-ends of one or both siNA strands.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) locked nucleicacid (LNA) nucleotides, for example, at the 5′-end, the 3′-end, both ofthe 5′ and 3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) 4′-thionucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In another embodiment, a siNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) acyclicnucleotides, for example, at the 5′-end, the 3′-end, both of the 5′ and3′-ends, or any combination thereof, of the siNA molecule.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises a sense strand or senseregion having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) 2′-O-alkyl (e.g., 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy,FANA, or abasic chemical modifications or any combination thereof.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises an antisense strand orantisense region having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30 or more) 2′-O-alkyl (e.g., 2′-O-methyl), 2′-deoxy-2′-fluoro,2′-deoxy, FANA, or abasic chemical modifications or any combinationthereof.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises a sense strand or senseregion and an antisense strand or antisense region, each having one ormore (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl(e.g., 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, FANA, or abasicchemical modifications or any combination thereof.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality(i.e. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are FANA pyrimidine nucleotides(e.g., wherein all pyrimidine nucleotides are FANA pyrimidinenucleotides or alternately a plurality (i.e. more than one) ofpyrimidine nucleotides are FANA pyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality(i.e. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region and an antisense region, wherein any (e.g., one or more orall) pyrimidine nucleotides present in the sense region and theantisense region are 2′-deoxy-2′-fluoro pyrimidine nucleotides (e.g.,wherein all pyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidinenucleotides or alternately a plurality (i.e. more than one) ofpyrimidine nucleotides are 2′-deoxy-2′-fluoro pyrimidine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) purine nucleotidespresent in the sense region are 2′-deoxy purine nucleotides (e.g.,wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality (i.e. more than one) of purine nucleotides are2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl purinenucleotides or alternately a plurality (i.e. more than one) ofpyrimidine nucleotides are 2-O-methyl purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoropyrimidine nucleotides (e.g., wherein all pyrimidine nucleotides are2′-deoxy-2′-fluoro pyrimidine nucleotides or alternately a plurality(i.e. more than one) of pyrimidine nucleotides are 2′-deoxy-2′-fluoropyrimidine nucleotides), and wherein any (e.g., one or more or all)purine nucleotides present in the sense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-deoxy purine nucleotides(e.g., wherein all purine nucleotides are 2′-deoxy purine nucleotides oralternately a plurality (i.e. more than one) of purine nucleotides are2′-deoxy purine nucleotides), wherein any nucleotides comprising a3′-terminal nucleotide overhang that are present in said sense regionare 2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the sense region are 2′-O-methyl purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality (i.e. more than one) of purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising asense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), wherein any (e.g., one or more or all) purine nucleotidespresent in the sense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides), and wherein any nucleotides comprising a 3′-terminalnucleotide overhang that are present in said sense region are 2′-deoxynucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), wherein any (e.g., one or more or all) purine nucleotidespresent in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides), and wherein any nucleotides comprising a 3′-terminalnucleotide overhang that are present in said antisense region are2′-deoxy nucleotides.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-deoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-deoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-deoxy purine nucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA) molecule of the invention comprising anantisense region, wherein any (e.g., one or more or all) pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and wherein any (e.g., one or more or all) purinenucleotides present in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides).

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid (siNA). molecule of the invention capable ofmediating RNA interference (RNAi) inside a cell or reconstituted invitro system comprising a sense region, wherein one or more pyrimidinenucleotides present in the sense region are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoroinethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and one or more purine nucleotides present in the senseregion are T-deoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-deoxy purine nucleotides or alternately a plurality(i.e. more than one) of purine nucleotides are T-deoxy purinenucleotides), and an antisense region, wherein one or more pyrimidinenucleotides present in the antisense region are 2′-deoxy-2′-fluoro,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy, pyrimidine nucleotides or alternately aplurality (i.e. more than one) of pyrimidine nucleotides are2′-deoxy-2′-fluoro, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy pyrimidinenucleotides), and one or more purine nucleotides present in theantisense region are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides (e.g., wherein all purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides or alternately aplurality (i.e. more than one) of purine nucleotides are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides). The sense region and/orthe antisense region can have a terminal cap modification, such as anymodification described herein or shown in FIG. 10, that is optionallypresent at the 3′-end, the 5′-end, or both of the 3′ and 5′-ends of thesense and/or antisense sequence. The sense and/or antisense region canoptionally further comprise a 3′-terminal nucleotide overhang havingabout 1 to about 4 (e.g., about 1, 2, 3, or 4) 2′-deoxynucleotides. Theoverhang nucleotides can further comprise one or more (e.g., about I, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages. Non-limiting examples ofthese chemically-modified siNAs are shown in FIGS. 4 and 5 and Table IIherein. In any of these described embodiments, the purine nucleotidespresent in the sense region are alternatively 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides) and one or morepurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides). Also, in any of these embodiments, one or more purinenucleotides present in the sense region are alternatively purineribonucleotides (e.g., wherein all purine nucleotides are purineribonucleotides or alternately a plurality (i.e. more than one) ofpurine nucleotides are purine ribonucleotides) and any purinenucleotides present in the antisense region are 2′-O-methyl, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides). Additionally, in any of these embodiments, one or morepurine nucleotides present in the sense region and/or present in theantisense region are alternatively selected from the group consisting of2′-deoxy nucleotides, locked nucleic acid (LNA) nucleotides,2′-methoxyethyl nucleotides, 4′-thionucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methyl nucleotides(e.g., wherein all purine nucleotides are selected from the groupconsisting of 2′-deoxy nucleotides, locked nucleic acid (LNA)nucleotides, 2′-methoxyethyl nucleotides, 4′-thionucleotides,2′-O-trifluoromethyl nucleotides, 2′-O-ethyl-trifluoromethoxynucleotides, 2′-O-difluoromethoxy-ethoxy nucleotides and 2′-O-methylnucleotides or alternately a plurality (i.e. more than one) of purinenucleotides are selected from the group consisting of 2′-deoxynucleotides, locked nucleic acid (LNA) nucleotides, 2′-methoxyethylnucleotides, 4′-thionucleotides, 2%0-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides and 2′-O-methyl nucleotides).

In another embodiment, any modified nucleotides present in the siNAmolecules of the invention, preferably in the antisense strand of thesiNA molecules of the invention, but also optionally in the sense and/orboth antisense and sense strands, comprise modified nucleotides havingproperties or characteristics similar to naturally occurringribonucleotides. For example, the invention features siNA moleculesincluding modified nucleotides having a Northern conformation (e.g.,Northern pseudorotation cycle, see for example Saenger, Principles ofNucleic Acid Structure, Springer-Verlag ed., 1984) otherwise known as a“ribo-like” or “A-form helix” configuration. As such, chemicallymodified nucleotides present in the siNA molecules of the invention,preferably in the antisense strand of the siNA molecules of theinvention, but also optionally in the sense and/or both antisense andsense strands, are resistant to nuclease degradation while at the sametime maintaining the capacity to mediate RNAi. Non-limiting examples ofnucleotides having a northern configuration include locked nucleic acid(LNA) nucleotides (e.g., 2′-O, 4′-C-methylene-(D-ribofuranosyl)nucleotides); 2′-methoxyethoxy (MOE) nucleotides; 2′methyl-thio-ethyl,2′-deoxy-2′-fluoro nucleotides, 2′-deoxy-2′-chloro nucleotides, 2′-azidonucleotides, 2′-O-trifluoromethyl nucleotides,2′-O-ethyl-trifluoromethoxy nucleotides, 2′-O-difluoromethoxy-ethoxynucleotides, 4′-thio nucleotides and 2′-O-methyl nucleotides.

In one embodiment, the sense strand of a double stranded siNA moleculeof the invention comprises a terminal cap moiety, (see for example FIG.10) such as an inverted deoxyabaisc moiety, at the 3′-end, 5′-end, orboth 3′ and 5′-ends of the sense strand.

In one embodiment, the invention features a chemically-modified shortinterfering nucleic acid molecule (siNA) capable of mediating RNAinterference (RNAi) inside a cell or reconstituted in vitro system,wherein the chemical modification comprises a conjugate covalentlyattached to the chemically-modified siNA molecule. Non-limiting examplesof conjugates contemplated by the invention include conjugates andligands described in Vargeese et al., U.S. Ser. No. 10/427,160, filedApr. 30, 2003, incorporated by reference herein in its entirety,including the drawings. In another embodiment, the conjugate iscovalently attached to the chemically-modified siNA molecule via abiodegradable linker. In one embodiment, the conjugate molecule isattached at the 3′-end of either the sense strand, the antisense strand,or both strands of the chemically-modified siNA molecule. In anotherembodiment, the conjugate molecule is attached at the 5′-end of eitherthe sense strand, the antisense strand, or both strands of thechemically-modified siNA molecule. In yet another embodiment, theconjugate molecule is attached both the 3′-end and 5′-end of either thesense strand, the antisense strand, or both strands of thechemically-modified siNA molecule, or any combination thereof. In oneembodiment, a conjugate molecule of the invention comprises a moleculethat facilitates delivery of a chemically-modified siNA molecule into abiological system, such as a cell. In another embodiment, the conjugatemolecule attached to the chemically-modified siNA molecule is a ligandfor a cellular receptor, such as peptides derived from naturallyoccurring protein ligands; protein localization sequences, includingcellular ZIP code sequences; antibodies; nucleic acid aptamers; vitaminsand other co-factors, such as folate and N-acetylgalactosamine;polymers, such as polyethyleneglycol (PEG); phospholipids; cholesterol;steroids, and polyamines, such as PEI, spennine or spermidine. Examplesof specific conjugate molecules contemplated by the instant inventionthat can be attached to chemically-modified siNA molecules are describedin Vargeese et al., U.S. Ser. No. 10/201,394, filed Jul. 22, 2002incorporated by reference herein. The type of conjugates used and theextent of conjugation of siNA molecules of the invention can beevaluated for improved pharmacokinetic profiles, bioavailability, and/orstability of siNA constructs while at the same time maintaining theability of the siNA to mediate RNAi activity. As such, one skilled inthe art can screen siNA constructs that are modified with variousconjugates to determine whether the siNA conjugate complex possessesimproved properties while maintaining the ability to mediate RNAi, forexample in animal models as are generally known in the art.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule of the invention, wherein the siNA furthercomprises a nucleotide, non-nucleotide, or mixednucleotide/non-nucleotide linker that joins the sense region of the siNAto the antisense region of the siNA. In one embodiment, a nucleotide,non-nucleotide, or mixed nucleotide/non-nucleotide linker is used, forexample, to attach a conjugate moiety to the siNA. In one embodiment, anucleotide linker of the invention can be a linker of >2 nucleotides inlength, for example about 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides inlength. In another embodiment, the nucleotide linker can be a nucleicacid aptamer. By “aptamer” or “nucleic acid aptamer” as used herein ismeant a nucleic acid molecule that binds specifically to a targetmolecule wherein the nucleic acid molecule has sequence that comprises asequence recognized by the target molecule in its natural setting.Alternately, an aptamer can be a nucleic acid molecule that binds to atarget molecule where the target molecule does not naturally bind to anucleic acid. The target molecule can be any molecule of interest. Forexample, the aptamer can be used to bind to a ligand-binding domain of aprotein, thereby preventing interaction of the naturally occurringligand with the protein. This is a non-limiting example and those in theart will recognize that other embodiments can be readily generated usingtechniques generally known in the art. (See, for example, Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Curr. Opin. Mol. Ther., 2, 100; Kusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628.)

In yet another embodiment, a non-nucleotide linker of the inventioncomprises abasic nucleotide, polyether, polyamine, polyamide, peptide,carbohydrate, lipid, polyhydrocarbon, or other polymeric compounds(e.g., polyethylene glycols such as those having between 2 and 100ethylene glycol units). Specific examples include those described bySeela and Kaiser, Nucleic Acids Res. 1990, 18:6353 and Nucleic AcidsRes. 1987, 15:3113; Cload and Schepartz, J. Am. Chem. Soc. 1991,113:6324; Richardson and Schepartz, J. Am. Chem. Soc. 1991, 113:5109; Maet al., Nucleic Acids Res. 1993, 21:2585 and Biochemistry 1993, 32:1751;Durand et al., Nucleic Acids Res. 1990, 18:6353; McCurdy et al.,Nucleosides & Nucleotides 1991, 10:287; Jschke et al., Tetrahedron Lett.1993, 34:301; Ono et al., Biochemistry 1991, 30:9914; Arnold et al.,International Publication No. WO 89/02439; Usman et al., InternationalPublication No. WO 95/06731; Dudycz et al., International PublicationNo. WO 95/11910 and Ferentz and Verdine, J. Am. Chem. Soc. 1991,113:4000, all hereby incorporated by reference herein. A“non-nucleotide” further means any group or compound that can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound can be abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine, for example at the C1 position of the sugar.

In one embodiment, the invention features a short interfering nucleicacid (siNA) molecule capable of mediating RNA interference (RNAi) insidea cell or reconstituted in vitro system, wherein one or both strands ofthe siNA molecule that are assembled from two separate oligonucleotidesdo not comprise any ribonucleotides. For example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA comprise separate oligonucleotides that do not haveany ribonucleotides (e.g., nucleotides having a 2′-OH group) present inthe oligonucleotides. In another example, a siNA molecule can beassembled from a single oligonculeotide where the sense and antisenseregions of the siNA are linked or circularized by a nucleotide ornon-nucleotide linker as described herein, wherein the oligonucleotidedoes not have any ribonucleotides (e.g., nucleotides having a 2′-OHgroup) present in the oligonucleotide. Applicant has surprisingly foundthat the presence of ribonucleotides (e.g., nucleotides having a2′-hydroxyl group) within the siNA molecule is not required or essentialto support RNAi activity. As such, in one embodiment, all positionswithin the siNA can include chemically modified nucleotides and/ornon-nucleotides such as nucleotides and or non-nucleotides havingFormula I, II, III, IV, V, VI, or VII or any combination thereof to theextent that the ability of the siNA molecule to support RNAi activity ina cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity in a cell or reconstituted invitro system comprising a single stranded polynucleotide havingcomplementarity to a target nucleic acid sequence. In anotherembodiment, the single stranded siNA molecule of the invention comprisesa 5′-terminal phosphate group. In another embodiment, the singlestranded siNA molecule of the invention comprises a 5′-terminalphosphate group and a 3′-terminal phosphate group (e.g., a 2′, 3′-cyclicphosphate). In another embodiment, the single stranded siNA molecule ofthe invention comprises about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides. Inyet another embodiment, the single stranded siNA molecule of theinvention comprises one or more chemically modified nucleotides ornon-nucleotides described herein. For example, all the positions withinthe siNA molecule can include chemically-modified nucleotides such asnucleotides having any of Formulae I-VII, or any combination thereof tothe extent that the ability of the siNA molecule to support RNAiactivity in a cell is maintained.

In one embodiment, a siNA molecule of the invention is a single strandedsiNA molecule that mediates RNAi activity or that alternately modulatesRNAi activity in a cell or reconstituted in vitro system comprising asingle stranded polynucleotide having complementarily to a targetnucleic acid sequence, wherein one or more pyrimidine nucleotidespresent in the siNA are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides (e.g., wherein allpyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides or alternately aplurality of pyrimidine nucleotides are 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifiuoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy pyrimidine nucleotides), and wherein anypurine nucleotides present in the antisense region are 2′-O-methyl,4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy, or2′-O-difluoromethoxy-ethoxy purine nucleotides (e.g., wherein all purinenucleotides are 2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl,2′-O-ethyl-trifluoromethoxy, or 2′-O-difluoromethoxy-ethoxy purinenucleotides or alternately a plurality of purine nucleotides are2′-O-methyl, 4′-thio, 2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,or 2′-O-difluoromethoxy-ethoxy purine nucleotides), and a terminal capmodification, such as any modification described herein or shown in FIG.10, that is optionally present at the 3′-end, the 5′-end, or both of the3′ and 5′-ends of the antisense sequence. The siNA optionally furthercomprises about 1 to about 4 or more (e.g., about 1, 2, 3, 4 or more)terminal 2′-deoxynucleotides at the 3′-end of the siNA molecule, whereinthe terminal nucleotides can further comprise one or more (e.g., 1, 2,3, 4 or more) phosphorothioate, phosphonoacetate, and/orthiophosphonoacetate internucleotide linkages, and wherein the siNAoptionally further comprises a terminal phosphate group, such as a5′-terminal phosphate group. In any of these embodiments, any purinenucleotides present in the antisense region are alternatively 2′-deoxypurine nucleotides (e.g., wherein all purine nucleotides are 2′-deoxypurine nucleotides or alternately a plurality of purine nucleotides are2′-deoxy purine nucleotides). Also, in any of these embodiments, anypurine nucleotides present in the siNA (i.e., purine nucleotides presentin the sense and/or antisense region) can alternatively be lockednucleic acid (LNA) nucleotides (e.g., wherein all purine nucleotides areLNA nucleotides or alternately a plurality of purine nucleotides are LNAnucleotides). Also, in any of these embodiments, any purine nucleotidespresent in the siNA are alternatively 2′-methoxyethyl purine nucleotides(e.g., wherein all purine nucleotides are 2′-methoxyethyl purinenucleotides or alternately a plurality of purine nucleotides are2′-methoxyethyl purine nucleotides). In another embodiment, any modifiednucleotides present in the single stranded siNA molecules of theinvention comprise modified nucleotides having properties orcharacteristics similar to naturally occurring ribonucleotides. Forexample, the invention features siNA molecules including modifiednucleotides having a Northern conformation (e.g., Northernpseudorotation cycle, see for example Saenger, Principles of NucleicAcid Structure, Springer-Verlag ed., 1984). As such, chemically modifiednucleotides present in the single stranded siNA molecules of theinvention are preferably resistant to nuclease degradation while at thesame time maintaining the capacity to mediate RNAi.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises a sense strand or senseregion having two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 ormore) 2′-O-alkyl (e.g., 2′-O-methyl) modifications or any combinationthereof. In another embodiment, the 2′-O-alkyl modification is atalternating position in the sense strand or sense region of the siNA,such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises an antisense strand orantisense region having two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, 30 or more) 2′-O-alkyl (e.g., 2′-O-methyl) modifications or anycombination thereof. In another embodiment, the 2′-O-alkyl modificationis at alternating position in the antisense strand or antisense regionof the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc.or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a chemically-modified short interfering nucleic acid(siNA) molecule of the invention comprises a sense strand or senseregion and an antisense strand or antisense region, each having two ormore (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) 2′-O-alkyl(e.g., 2′-O-methyl), 2′-deoxy-2′-fluoro, 2′-deoxy, or abasic chemicalmodifications or any combination thereof. In another embodiment, the2′-O-alkyl modification is at alternating position in the sense strandor sense region of the siNA, such as position 1, 3, 5, 7, 9, 11, 13, 15,17, 19, 21 etc. or position 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 etc. Inanother embodiment, the 2′-O-alkyl modification is at alternatingposition in the antisense strand or antisense region of the siNA, suchas position 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 etc. or position 2, 4,6, 8, 10, 12, 14, 16, 18, 20 etc.

In one embodiment, a siNA molecule of the invention comprises chemicallymodified nucleotides or non-nucleotides (e.g., having any of FormulaeI-VII, such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides) at alternatingpositions within one or more strands or regions of the siNA molecule.For example, such chemical modifications can be introduced at everyother position of a RNA based siNA molecule, starting at either thefirst or second nucleotide from the 3′-end or 5′-end of the siNA. In anon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 of eachstrand are chemically modified (e.g., with compounds having any ofFormulae I-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In anothernon-limiting example, a double stranded siNA molecule of the inventionin which each strand of the siNA is 21 nucleotides in length is featuredwherein positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20 of each strandare chemically modified (e.g., with compounds having any of FormulaeI-VII, such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides). In oneembodiment, one strand of the double stranded siNA molecule compriseschemical modifications at positions 2, 4, 6, 8, 10, 12, 14, 16, 18, and20 and chemical modifications at positions 1, 3, 5, 7, 9, 11, 13, 15,17, 19 and 21. Such siNA molecules can further comprise terminal capmoieties and/or backbone modifications as described herein.

In one embodiment, a siNA molecule of the invention comprises thefollowing features: if purine nucleotides are present at the 5′-end(e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 fromthe 5′-end) of the antisense strand or antisense region (otherwisereferred to as the guide sequence or guide strand) of the siNA moleculethen such purine nucleosides are ribonucleotides. In another embodiment,the purine ribonucleotides, when present, are base paired to nucleotidesof the sense strand or sense region (otherwise referred to as thepassenger strand) of the siNA molecule. Such purine ribonucleotides canbe present in a siNA stabilization motif that otherwise comprisesmodified nucleotides.

In one embodiment, a siNA molecule of the invention comprises thefollowing features: if pyrimidine nucleotides are present at the 5′-end(e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 fromthe 5′-end) of the antisense strand or antisense region (otherwisereferred to as the guide sequence or guide strand) of the siNA moleculethen such pyrimidine nucleosides are ribonucleotides. In anotherembodiment, the pyrimidine ribonucleotides, when present, are basepaired to nucleotides of the sense strand or sense region (otherwisereferred to as the passenger strand) of the siNA molecule. Suchpyrimidine ribonucleotides can be present in a siNA stabilization motifthat otherwise comprises modified nucleotides.

In one embodiment, a siNA molecule of the invention comprises thefollowing features: if pyrimidine nucleotides are present at the 5′-end(e.g., at any of terminal nucleotide positions 1, 2, 3, 4, 5, or 6 fromthe 5′-end) of the antisense strand or antisense region (otherwisereferred to as the guide sequence or guide strand) of the siNA moleculethen such pyrimidine nucleosides are modified nucleotides. In anotherembodiment, the modified pyrimidine nucleotides, when present, are basepaired to nucleotides of the sense strand or sense region (otherwisereferred to as the passenger strand) of the siNA molecule. Non-limitingexamples of modified pyrimidine nucleotides include those having any ofFormulae I-VIII such as such as 2′-deoxy, 2′-deoxy-2′-fluoro, 4′-thio,2′-O-trifluoromethyl, 2′-O-ethyl-trifluoromethoxy,2′-O-difluoromethoxy-ethoxy or 2′-O-methyl nucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SI:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions wherein any purine nucleotides when present are        ribonueleotides; X1 and X2 are independently integers from about        0 to about 4; X3 is an integer from about 9 to about 30; X4 is        an integer from about 11 to about 30, provided that the sum of        X4 and X5 is between 17-36; X5 is an integer from about 1 to        about 6; NX3 is complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a    combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′ deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are independently    2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of    2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions wherein any purine nucleotides when present are        ribonucleotides; X1 and X2 are independently integers from about        0 to about 4; X3 is an integer from about 9 to about 30; X4 is        an integer from about 11 to about 30, provided that the sum of        X4 and X5 is between 17-36; X5 is an integer from about 1 to        about 6; NX3 is complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are ribonucleotides; any purine nucleotides present in the    sense strand (upper strand) are ribonucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SIII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions wherein any purine nucleotides when present are        ribonucleotides; X1 and X2 are independently integers from about        0 to about 4; X3 is an integer from about 9 to about 30; X4 is        an integer from about 11 to about 30, provided that the sum of        X4 and X5 is between 17-36; X5 is an integer from about 1 to        about 6; NX3 is complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are ribonucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SIV:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions wherein any purine nucleotides when present are        ribonucleotides; X1 and X2 are independently integers from about        0 to about 4; X3 is an integer from about 9 to about 30; X4 is        an integer from about 11 to about 30, provided that the sum of        X4 and X5 is between 17-36; X5 is an integer from about 1 to        about 6; NX3 is complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are deoxyribonucleotides;    and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SV:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions wherein any purine nucleotides when present are        ribonucleotides; X1 and X2 are independently integers from about        0 to about 4; X3 is an integer from about 9 to about 30; X4 is        an integer from about 11 to about 30, provided that the sum of        X4 and X5 is between 17-36; X5 is an integer from about 1 to        about 6; NX3 is complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are nucleotides having a ribo-like configuration    (e.g., Northern or A-form helix configuration); any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are nucleotides having a ribo-like configuration (e.g.,    Northern or A-form helix configuration); any purine nucleotides    present in the sense strand (upper strand) are 2′-O-methyl    nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SVI:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions comprising sequence that renders the 5′-end of the        antisense strand (lower strand) less thermally stable than the        5′-end of the sense strand (upper strand); X1 and X2 are        independently integers from about 0 to about 4; X3 is an integer        from about 9 to about 30; X4 is an integer from about 11 to        about 30, provided that the sum of X4 and X5 is between 17-36;        X5 is an integer from about 1 to about 6; NX3 is complementary        to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a    combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are independently    deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of    2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SVII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides; X1 and X2        are independently integers from about 0 to about 4; X3 is an        integer from about 9 to about 30; X4 is an integer from about 11        to about 30; NX3 is complementary to NX4, and any (N)        nucleotides are 2′-O-methyl and/or 2′-deoxy-2′-fluoro        nucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SVIII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions comprising sequence that renders the 5′-end of the        antisense strand (lower strand) less thermally stable than the        5′-end of the sense strand (upper strand); [N] represents        nucleotide positions that are ribonucleotides; X1 and X2 are        independently integers from about 0 to about 4; X3 is an integer        from about 9 to about 15; X4 is an integer from about 11 to        about 30, provided that the sum of X4 and X5 is between 17-36;        X5 is an integer from about 1 to about 6; X6 is an integer from        about 1 to about 4; X7 is an integer from about 9 to about 15;        NX7, NX6, and NX3 are complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a    combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′ fluoro nucleotides other than [N]    nucleotides; any purine nucleotides present in the sense strand    (upper strand) are independently 2′-deoxyribonucleotides,    2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides    and 2′-O-methyl nucleotides other than [N] nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SIX:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 30; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; NX3 is complementary to NX4 and        NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a    combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are independently    2′-deoxyribonucleotides, 2′-O-methyl nucleotides or a combination of    2′-deoxyribonucleotides and 2′-O-methyl nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SX:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 30; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; NX3 is complementary to NX4 and        NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are ribonucleotides; any purine nucleotides present in the    sense strand (upper strand) are ribonucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SXI:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 30; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; NX3 is complementary to NX4 and        NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are ribonucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SXII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 30; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; NX3 is complementary to NX4 and        NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides; any purine nucleotides    present in the sense strand (upper strand) are deoxyribonucleotides;    and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SXIII:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 30; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; NX3 is complementary to NX4 and        NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are nucleotides having a ribo-like configuration    (e.g., Northern or A-form helix configuration); any purine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are nucleotides having a ribo-like configuration (e.g.,    Northern or A-fonn helix configuration); any purine nucleotides    present in the sense strand (upper strand) are 2′-O-methyl    nucleotides;

and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, the invention features a double stranded nucleic acidmolecule having structure SXIV:

-   -   wherein each N is independently a nucleotide; each B is a        terminal cap moiety that can be present or absent; (N)        represents non-base paired or overhanging nucleotides which can        be unmodified or chemically modified; [N] represents nucleotide        positions that are ribonucleotides; [N] represents nucleotide        positions that are ribonucleotides; X1 and X2 are independently        integers from about 0 to about 4; X3 is an integer from about 9        to about 15; X4 is an integer from about 11 to about 30,        provided that the sum of X4 and X5 is between 17-36; X5 is an        integer from about 1 to about 6; X6 is an integer from about 1        to about 4; X7 is an integer from about 9 to about 15; NX7, NX6,        and NX3 are complementary to NX4 and NX5, and

-   (a) any pyrimidine nucleotides present in the antisense strand    (lower strand) are 2′-deoxy-2′-fluoro nucleotides; any patine    nucleotides present in the antisense strand (lower strand) other    than the purines nucleotides in the [N] nucleotide positions, are    independently 2′-O-methyl nucleotides, 2′-deoxyribonucleotides or a    combination of 2′-deoxyribonucleotides and 2′-O-methyl nucleotides;

-   (b) any pyrimidine nucleotides present in the sense strand (upper    strand) are 2′-deoxy-2′-fluoro nucleotides other than [N]    nucleotides; any purine nucleotides present in the sense strand    (upper strand) are independently 2′-deoxyribonucleotides,    2′-O-methyl nucleotides or a combination of 2′-deoxyribonucleotides    and 2′-O-methyl nucleotides other than [N] nucleotides; and

-   (c) any (N) nucleotides are optionally 2′-O-methyl,    2′-deoxy-2′-fluoro, or deoxyribonucleotides.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises a terminal phosphate group at the 5′-end of theantisense strand or antisense region of the nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV comprises X5=1, 2, or 3; each X1 and X2=1 or 2; X3=12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or30, and X4=15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29,or 30.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises X5=1; each X1 and X2=2; X3=19, and X4=18.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SIT, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises X5=2; each X1 and X2=2; X3=19, and X4=17

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV comprises X5=3; each X1 and X2=2; X3=19, and X4=16.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV comprises B at the 3′ and 5′ ends of the sense strand orsense region.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV comprises B at the 3′-end of the antisense strand orantisense region.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV comprises B at the 3′ and 5′ ends of the sense strand orsense region and B at the 3′-end of the antisense strand or antisenseregion.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIII, SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII,SXIII, or SXIV further comprises one or more phosphorothioateinternucleotide linkages at the first terminal (N) on the 3′end of thesense strand, antisense strand, or both sense strand and antisensestrands of the nucleic acid molecule. For example, a double strandednucleic acid molecule can comprise X1 and/or X2=2 having overhangingnucleotide positions with a phosphorothioate internucleotide linkage,e.g., (NsN) where “s” indicates phosphorothioate.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises (N) nucleotides that are 2′-O-methyl nucleotides.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises (N) nucleotides that are 2′-deoxy nucleotides.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises (N) nucleotides in the antisense strand (lower strand)that are complementary to nucleotides in a target polynucleotidesequence having complementary to the N and [N] nucleotides of theantisense (lower) strand.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises (N) nucleotides in the sense strand (upper strand)that comprise a contiguous nucleotide sequence of about 15 to about 30nucleotides of a target polynucleotide sequence.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV comprises (N) nucleotides in the sense strand (upper strand)that comprise nucleotide sequence corresponding a target polynucleotidesequence having complementary to the antisense (lower) strand such thatthe contiguous (N) and N nucleotide sequence of the sense strandcomprises nucleotide sequence of the target nucleic acid sequence.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SVIII or SXIV comprises B only at the 5′-end of the sense(upper) strand of the double stranded nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SI, SII, SIR SIV, SV, SVI, SVII, SVIII, SIX, SX, SXII, SXIII,or SXIV further comprises an unpaired terminal nucleotide at the 5′-endof the antisense (lower) strand. The unpaired nucleotide is notcomplementary to the sense (upper) strand. In one embodiment, theunpaired terminal nucleotide is complementary to a target polynucleotidesequence having complementary to the N and [N] nucleotides of theantisense (lower) strand. In another embodiment, the unpaired terminalnucleotide is not complementary to a target polynucleotide sequencehaving complementary to the N and [N] nucleotides of the antisense(lower) strand.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SVIII or SXIV comprises X6=1 and X3=10.

In one embodiment, a double stranded nucleic acid molecule having any ofstructure SVIII or SXIV comprises X6=2 and X3=9.

In one embodiment, the invention features a composition comprising asiNA molecule or double stranded nucleic acid molecule or RNAi inhibitorformulated as any of formulation LNP-051; LNP-053; LNP-054; LNP-069;LNP-073; LNP-077; LNP-080; LNP 082; LNP-083; LNP-060; LNP-061; LNP-086;LNP-097; LNP-098; LNP-099; LNP-100; LNP-101; LNP-102; LNP-103; orLNP-104 (see Table IV).

In one embodiment, the invention features a composition comprising afirst double stranded nucleic and a second double stranded nucleic acidmolecule each having a first strand and a second strand that arecomplementary to each other, wherein the second strand of the firstdouble stranded nucleic acid molecule comprises sequence complementaryto a first target sequence and the second strand of the second doublestranded nucleic acid molecule comprises sequence complementary to asecond target or pathway target sequence. In one embodiment, thecomposition further comprises a cationic lipid, a neutral lipid, and apolyethyleneglycol-conjugate. In one embodiment, the composition furthercomprises a cationic lipid, a neutral lipid, apolyethyleneglycol-conjugate, and a cholesterol. In one embodiment, thecomposition further comprises a polyethyleneglycol-conjugate, acholesterol, and a surfactant. In one embodiment, the cationic lipid isselected from the group consisting of CLinDMA, pCLinDMA, eCLinDMA,DMOBA, and DMLBA. In one embodiment, the neutral lipid is selected fromthe group consisting of DSPC, DOBA, and cholesterol. In one embodiment,the polyethyleneglycol-conjugate is selected from the group consistingof a PEG-dimyristoyl glycerol and PEG-cholesterol. In one embodiment,the PEG is 2KPEG. In one embodiment, the surfactant is selected from thegroup consisting of palmityl alcohol, stearyl alcohol, oleyl alcohol andlinoleyl alcohol. In one embodiment, the cationic lipid is CLinDMA, theneutral lipid is DSPC, the polyethylene glycol conjugate is 2KPEG-DMG,the cholesterol is cholesterol, and the surfactant is linoleyl alcohol.In one embodiment, the CLinDMA, the DSPC, the 2KPEG-DMG, thecholesterol, and the linoleyl alcohol are present in molar ratio of43:38:10:2:7 respectively.

In any of the embodiments herein, the siNA molecule of the inventionmodulates expression of one or more targets via RNA interference or theinhibition of RNA interference. In one embodiment, the RNA interferenceis RISC mediated cleavage of the target (e.g., siRNA mediated RNAinterference). In one embodiment, the RNA interference is translationalinhibition of the target (e.g., miRNA mediated RNA interference). In oneembodiment, the RNA interference is transcriptional inhibition of thetarget (e.g., siRNA mediated transcriptional silencing). In oneembodiment, the RNA interference takes place in the cytoplasm. In oneembodiment, the RNA interference takes place in the nucleus.

In any of the embodiments herein, the siNA molecule of the inventionmodulates expression of one or more targets via inhibition of anendogenous target RNA, such as an endogenous mRNA, siRNA, miRNA, oralternately though inhibition of RISC.

In one embodiment, the invention features one or more RNAi inhibitorsthat modulate the expression of one or more gene targets by miRNAinhibition, siRNA inhibition, or RISC inhibition.

In one embodiment, a RNAi inhibitor of the invention is a siNA moleculeas described herein that has one or more strands that are complementaryto one or more target miRNA or siRNA molecules.

In one embodiment, the RNAi inhibitor of the invention is an antisensemolecule that is complementary to a target miRNA or siRNA molecule or aportion thereof. An antisense RNAi inhibitor of the invention can be oflength of about 10 to about 40 nucleotides in length (e.g., 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides in length). Anantisense RNAi inhibitor of the invention can comprise one or moremodified nucleotides or non-nucleotides as described herein (see forexample molecules having any of Formulae I-VII herein or any combinationthereof). In one embodiment, an antisense RNAi inhibitor of theinvention can comprise one or more or all 2′-O-methyl nucleotides. Inone embodiment, an antisense RNAi inhibitor of the invention cancomprise one or more or all 2′-deoxy-2′-fluoro nucleotides. In oneembodiment, an antisense RNAi inhibitor of the invention can compriseone or more or all 2′-O-methoxy-ethyl (also known as 2′-methoxyethoxy orMOE) nucleotides. In one embodiment, an antisense RNAi inhibitor of theinvention can comprise one or more or all phosphorothioateinternucleotide linkages. In one embodiment, an antisense RNA inhibitoror the invention can comprise a terminal cap moiety at the 3′-end, the5′-end, or both the 5′ and 3′ ends of the antisense RNA inhibitor.

In one embodiment, a RNAi inhibitor of the invention is a nucleic acidaptamer having binding affinity for RISC, such as a regulatable aptamer(see for example An et al., 2006, RNA, 12:710-716). An aptamer RNAiinhibitor of the invention can be of length of about 10 to about 50nucleotides in length (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides inlength). An aptamer RNAi inhibitor of the invention can comprise one ormore modified nucleotides or non-nucleotides as described herein (seefor example molecules having any of Formulae I-VII herein or anycombination thereof). In one embodiment, an aptamer RNAi inhibitor ofthe invention can comprise one or more or all 2′-O-methyl nucleotides.In one embodiment, an aptamer RNAi inhibitor of the invention cancomprise one or more or all 2′-deoxy-2′-fluoro nucleotides. In oneembodiment, an aptamer RNAi inhibitor of the invention can comprise oneor more or all 2′-O-methoxy-ethyl (also known as 2′-methoxyethoxy orMOE) nucleotides. In one embodiment, an aptamer RNAi inhibitor of theinvention can comprise one or more or all phosphorothioateinternucleotide linkages. In one embodiment, an aptamer RNA inhibitor orthe invention can comprise a terminal cap moiety at the 3′-end, the5′-end, or both the 5′ and 3′ ends of the aptamer RNA inhibitor.

In one embodiment, the invention features a method for modulating theexpression of a target gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified orunmodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the target gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the target gene in the cell.

In one embodiment, the invention features a method for modulating theexpression of a target gene within a cell comprising: (a) synthesizing asiNA molecule of the invention, which can be chemically-modified orunmodified, wherein one of the siNA strands comprises a sequencecomplementary to RNA of the target gene and wherein the sense strandsequence of the siNA comprises a sequence identical or substantiallysimilar to the sequence of the target RNA; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the target gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one target gene within a cell comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified or unmodified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the target genes; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of two or more target genes within a cell comprising: (a)synthesizing one or more siNA molecules of the invention, which can bechemically-modified or unmodified, wherein the siNA strands comprisesequences complementary to RNA of the target genes and wherein the sensestrand sequences of the siNAs comprise sequences identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecules into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one target gene within a cell comprising:(a) synthesizing a siNA molecule of the invention, which can bechemically-modified or unmodified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the target gene and whereinthe sense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNAs; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the target genes in the cell.

In another embodiment, the invention features a method for modulatingthe expression of a target gene within a cell comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified or unmodified, wherein one of the siNA strandscomprises a sequence complementary to RNA of the target gene, whereinthe sense strand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequences of the target RNA; and (b)introducing the siNA molecule into a cell under conditions suitable tomodulate (e.g., inhibit) the expression of the target gene in the cell.

In one embodiment, siNA molecules of the invention are used as reagentsin ex vivo applications. For example, siNA reagents are introduced intotissue or cells that are transplanted into a subject for therapeuticeffect. The cells and/or tissue can be derived from an organism orsubject that later receives the explant, or can be derived from anotherorganism or subject prior to transplantation. The siNA molecules can beused to modulate the expression of one or more genes in the cells ortissue, such that the cells or tissue obtain a desired phenotype or areable to perform a function when transplanted in vivo. In one embodiment,certain target cells from a patient are extracted. These extracted cellsare contacted with siNAs targeting a specific nucleotide sequence withinthe cells under conditions suitable for uptake of the siNAs by thesecells (e.g., using delivery reagents such as cationic lipids, liposomesand the like or using techniques such as electroporation to facilitatethe delivery of siNAs into cells). The cells are then reintroduced backinto the same patient or other patients.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the target gene; and (b) introducingthe siNA molecule into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the target gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the target gene in that organism.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a tissue explant comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the target gene and wherein the sensestrand sequence of the siNA comprises a sequence identical orsubstantially similar to the sequence of the target RNA; and (b)introducing the siNA molecule into a cell of the tissue explant derivedfrom a particular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the target gene in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the target gene in that organism.

In another embodiment, the invention features a method of modulating theexpression of more than one target gene in a tissue explant comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the target genes; and (b) introducingthe siNA molecules into a cell of the tissue explant derived from aparticular organism under conditions suitable to modulate (e.g.,inhibit) the expression of the target genes in the tissue explant. Inanother embodiment, the method further comprises introducing the tissueexplant back into the organism the tissue was derived from or intoanother organism under conditions suitable to modulate (e.g., inhibit)the expression of the target genes in that organism.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the target gene; and (b) introducingthe siNA molecule into the subject or organism under conditions suitableto modulate (e.g., inhibit) the expression of the target gene in thesubject or organism. The level of target protein or RNA can bedetermined using various methods well-known in the art.

In another embodiment, the invention features a method of modulating theexpression of more than one target gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein one of the siNA strands comprises asequence complementary to RNA of the target genes; and (b) introducingthe siNA molecules into the subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the target genesin the subject or organism. The level of target protein or RNA can bedetermined as is known in the art.

In one embodiment, the invention features a method for modulating theexpression of a target gene within a cell, comprising: (a) synthesizinga siNA molecule of the invention, which can be chemically-modified,wherein the siNA comprises a single stranded sequence havingcomplementarity to RNA of the target gene; and (b) introducing the siNAmolecule into a cell under conditions suitable to modulate (e.g.,inhibit) the expression of the target gene in the cell.

In another embodiment, the invention features a method for modulatingthe expression of more than one target gene within a cell, comprising:(a) synthesizing siNA molecules of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarily to RNA of the target gene; and (b)contacting the cell in vitro or in vivo with the siNA molecule underconditions suitable to modulate (e.g., inhibit) the expression of thetarget genes in the cell.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a tissue explant ((e.g., any organ,tissue or cell as can be transplanted from one organism to another orback to the same organism from which the organ, tissue or cell isderived) comprising: (a) synthesizing a siNA molecule of the invention,which can be chemically-modified, wherein the siNA comprises a singlestranded sequence having complementarity to RNA of the target gene; and(b) contacting a cell of the tissue explant derived from a particularsubject or organism with the siNA molecule under conditions suitable tomodulate (e.g., inhibit) the expression of the target gene in the tissueexplant. In another embodiment, the method further comprises introducingthe tissue explant back into the subject or organism the tissue wasderived from or into another subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the target genein that subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one target gene in a tissue explant (e.g., anyorgan, tissue or cell as can be transplanted from one organism toanother or back to the same organism from which the organ, tissue orcell is derived) comprising: (a) synthesizing siNA molecules of theinvention, which can be chemically-modified, wherein the siNA comprisesa single stranded sequence having complementarity to RNA of the targetgene; and (b) introducing the siNA molecules into a cell of the tissueexplant derived from a particular subject or organism under conditionssuitable to modulate (e.g., inhibit) the expression of the target genesin the tissue explant. In another embodiment, the method furthercomprises introducing the tissue explant back into the subject ororganism the tissue was derived from or into another subject or organismunder conditions suitable to modulate (e.g., inhibit) the expression ofthe target genes in that subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a subject or organism comprising: (a)synthesizing a siNA molecule of the invention, which can bechemically-modified, wherein the siNA comprises a single strandedsequence having complementarily to RNA of the target gene; and (b)introducing the siNA molecule into the subject or organism underconditions suitable to modulate (e.g., inhibit) the expression of thetarget gene in the subject or organism.

In another embodiment, the invention features a method of modulating theexpression of more than one target gene in a subject or organismcomprising: (a) synthesizing siNA molecules of the invention, which canbe chemically-modified, wherein the siNA comprises a single strandedsequence having complementarity to RNA of the target gene; and (b)introducing the siNA molecules into the subject or organism underconditions suitable to modulate (e.g., inhibit) the expression of thetarget genes in the subject or organism.

In one embodiment, the invention features a method of modulating theexpression of a target gene in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate (e.g., inhibit) the expression ofthe target gene in the subject or organism.

In one embodiment, the invention features a method for treating orpreventing a disease, disorder, trait or condition related to geneexpression or activity in a subject or organism comprising contactingthe subject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the target gene in thesubject or organism. The reduction of gene expression and thus reductionin the level of the respective protein/RNA relieves, to some extent, thesymptoms of the disease, disorder, trait or condition.

In one embodiment, the invention features a method for treating orpreventing cancer in a subject or organism comprising contacting thesubject or organism with a siNA molecule of the invention underconditions suitable to modulate the expression of the target gene in thesubject or organism whereby the treatment or prevention of cancer can beachieved. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via localadministration to relevant tissues or cells, such as cancerous cells andtissues. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of cancer in a subject or organism.The siNA molecule of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism. The siNA molecule can becombined with other therapeutic treatments and modalities as are knownin the art for the treatment of or prevention of cancer in a subject ororganism.

In one embodiment, the invention features a method for treating orpreventing a proliferative disease or condition in a subject or organismcomprising contacting the subject or organism with a siNA molecule ofthe invention under conditions suitable to modulate the expression ofthe target gene in the subject or organism whereby the treatment orprevention of the proliferative disease or condition can be achieved. Inone embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via local administrationto relevant tissues or cells, such as cells and tissues involved inproliferative disease. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia systemic administration (such as via intravenous or subcutaneousadministration of siNA) to relevant tissues or cells, such as tissues orcells involved in the maintenance or development of the proliferativedisease or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism: The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of proliferative diseases, traits, disorders,or conditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing transplant and/or tissue rejection (allograft rejection) in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of transplant and/or tissue rejection (allograftrejection) can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in transplant and/or tissue rejection (allograftrejection). In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of transplant and/or tissue rejection(allograft rejection) in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of transplant and/or tissue rejection(allograft rejection) in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing an autoimmune disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the autoimmune disease, disorder, trait orcondition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the autoimmune disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the autoimmune disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of autoimmune diseases, traits, disorders, orconditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing an infectious disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the infectious disease, disorder, trait orcondition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the infectious disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the infectious disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of infectious diseases, traits, or conditionsin a subject or organism.

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil in combination with asiNA molecule of the invention; wherein the Adefovir Dipivoxil and thesiNA molecule are administered under conditions suitable for reducing orinhibiting the level of Hepatitis B Virus (HBV) in the subject comparedto a subject not treated with the Adefovir Dipivoxil and the siNAmolecule. In one embodiment, a siNA molecule of the invention isformulated as a composition described in U.S. Provisional patentapplication No. 60/678,531 and in related U.S. Provisional patentapplication No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisionalpatent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese etal.), all of which are incorporated by reference herein in theirentirety. Such siNA formulations are generally referred to as “lipidnucleic acid particles” (LNP).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Lamivudine (3TC) in combination with a siNAmolecule of the invention; wherein the Lamivudine (3TC) and the siNA areadministered under conditions suitable for reducing or inhibiting thelevel of Hepatitis B Virus (HBV) in the subject compared to a subjectnot treated with the Lamivudine (3TC) and the siNA molecule. In oneembodiment, the siNA molecule or double stranded nucleic acid moleculeof the invention is formulated as a composition described in U.S.Provisional patent application No. 60/678,531 and in related U.S.Provisional patent application No. 60/703,946, filed Jul. 29, 2005, andU.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005(Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil and Lamivudine (3TC) incombination with a siNA molecule of the invention; wherein the AdefovirDipivoxil and Lamivudine (3TC) and the siNA molecule are administeredunder conditions suitable for reducing or inhibiting the level ofHepatitis B Virus (HBV) in the subject compared to a subject not treatedwith the Adefovir Dipivoxil and Lamivudine (3TC) and the siNA molecule.In one embodiment, the siNA molecule or double stranded nucleic acidmolecule of the invention is formulated as a composition described inU.S. Provisional patent application No. 60/678,531 and in related U.S.Provisional patent application No. 60/703,946, filed Jul. 29, 2005, andU.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005(Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense strand are complementary to the antisensestrand; (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;and wherein the Adefovir Dipivoxil and the double stranded nucleic acidmolecule are administered under conditions suitable for reducing orinhibiting the level of Hepatitis B Virus (HBV) in the subject comparedto a subject not treated with the Adefovir Dipivoxil and the doublestranded nucleic acid molecule. In one embodiment, the siNA molecule ordouble stranded nucleic acid molecule of the invention is formulated asa composition described in U.S. Provisional patent application No.60/678,531 and in related U.S. Provisional patent application No.60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent applicationNo. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Lamivudine (3TC) in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense strand are complementary to the antisensestrand; (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;and wherein the Lamivudine (3TC) and the double stranded nucleic acidmolecule are administered under conditions suitable for reducing orinhibiting the level of Hepatitis B Virus (HBV) in the subject comparedto a subject not treated with the Lamivudine (3TC) and the doublestranded nucleic acid molecule. In one embodiment, the siNA molecule ordouble stranded nucleic acid molecule of the invention is formulated asa composition described in U.S. Provisional patent application No.60/678,531 and in related U.S. Provisional patent application No.60/703,946, filed Jul. 29, 2005, and U.S. Provisional patent applicationNo. 60/737,024, filed Nov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil and Lamivudine (3TC) incombination with a chemically synthesized double stranded nucleic acidmolecule; wherein (a) the double stranded nucleic acid moleculecomprises a sense strand and an antisense strand; (b) each strand of thedouble stranded nucleic acid molecule is 15 to 28 nucleotides in length;(c) at least 15 nucleotides of the sense strand are complementary to theantisense strand; (d) the antisense strand of the double strandednucleic acid molecule has complementarity to a Hepatitis B Virus (HBV)target RNA; and wherein the Adefovir Dipivoxil and Lamivudine (3TC) andthe double stranded nucleic acid molecule are administered underconditions suitable for reducing or inhibiting the level of Hepatitis BVirus (HBV) in the subject compared to a subject not treated with theAdefovir Dipivoxil and Lamivudine (3TC) and the double stranded nucleicacid molecule. In one embodiment, the siNA molecule or double strandednucleic acid molecule of the invention is formulated as a compositiondescribed in U.S. Provisional patent application No. 60/678,531 and inrelated U.S. Provisional patent application No. 60/703,946, filed Jul.29, 2005, and U.S. Provisional patent application No. 60/737,024, filedNov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense strand are complementary to the antisensestrand; (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;(e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having achemical modification; and (f) at least two of the chemicalmodifications are different from each other, and wherein the AdefovirDipivoxil and the double stranded nucleic acid molecule are administeredunder conditions suitable for reducing or inhibiting the level ofHepatitis B Virus (HBV) in the subject compared to a subject not treatedwith the Adefovir Dipivoxil and the double stranded nucleic acidmolecule. In one embodiment, the siNA molecule or double strandednucleic acid molecule of the invention is formulated as a compositiondescribed in U.S. Provisional patent application No. 60/678,531 and inrelated U.S. Provisional patent application No. 60/703,946, filed Jul.29, 2005, and U.S. Provisional patent application No. 60/737,024, filedNov. 15, 2005 (Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Lamivudine (3TC) in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense strand are complementary to the antisensestrand (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;(e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having achemical modification; and (f) at least two of the chemicalmodifications are different from each other, and wherein the Lamivudine(3TC) and the double stranded nucleic acid molecule are administeredunder conditions suitable for reducing or inhibiting the level ofHepatitis B Virus (HBV) in the subject compared to a subject not treatedwith the Lamivudine (3TC) and the double stranded nucleic acid molecule.In one embodiment, the siNA molecule or double stranded nucleic acidmolecule of the invention is formulated as a composition described inU.S. Provisional patent application No. 60/678,531 and in related U.S.Provisional patent application No. 60/703,946, filed Jul. 29, 2005, andU.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005(Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil and Lamivudine (3TC) incombination with a chemically synthesized double stranded nucleic acidmolecule; wherein (a) the double stranded nucleic acid moleculecomprises a sense strand and an antisense strand; (b) each strand of thedouble stranded nucleic acid molecule is 15 to 28 nucleotides in length;(c) at least 15 nucleotides of the sense strand are complementary to theantisense strand (d) the antisense strand of the double stranded nucleicacid molecule has complementarity to a Hepatitis B Virus (HBV) targetRNA; (e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having achemical modification; and (f) at least two of the chemicalmodifications are different from each other, and wherein the AdefovirDipivoxil and Lamivudine (3TC) and the double stranded nucleic acidmolecule are administered under conditions suitable for reducing orinhibiting the level of Hepatitis B Virus (HBV) in the subject comparedto a subject not treated with the Adefovir Dipivoxil and Lamivudine(3TC) and the double stranded nucleic acid molecule. In one embodiment,the siNA molecule or double stranded nucleic acid molecule of theinvention is formulated as a composition described in U.S. Provisionalpatent application No. 60/678,531 and in related U.S. Provisional patentapplication No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisionalpatent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese etal.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense, strand are complementary to the antisensestrand; (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;(e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having asugar modification; and (f) at least two of the sugar modifications aredifferent from each other, and wherein the Adefovir Dipivoxil and thedouble stranded nucleic acid molecule are administered under conditionssuitable for reducing or inhibiting the level of Hepatitis B Virus (HBV)in the subject compared to a subject not treated with the AdefovirDipivoxil and the double stranded nucleic acid molecule. In oneembodiment, the siNA molecule or double stranded nucleic acid moleculeof the invention is formulated as a composition described in U.S.Provisional patent application No. 60/678,531 and in related U.S.Provisional patent application No. 60/703,946, filed Jul. 29, 2005, andU.S. Provisional patent application No. 60/737,024, filed Nov. 15, 2005(Vargeese et al.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Lamivudine (3TC) in combination with achemically synthesized double stranded nucleic acid molecule; wherein(a) the double stranded nucleic acid molecule comprises a sense strandand an antisense strand; (b) each strand of the double stranded nucleicacid molecule is 15 to 28 nucleotides in length; (c) at least 15nucleotides of the sense strand are complementary to the antisensestrand (d) the antisense strand of the double stranded nucleic acidmolecule has complementarity to a Hepatitis B Virus (HBV) target RNA;(e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having asugar modification; and (f) at least two of the sugar modifications aredifferent from each other, and wherein the Lamivudine (3TC) and thedouble stranded nucleic acid molecule are administered under conditionssuitable for reducing or inhibiting the level of Hepatitis B Virus (HBV)in the subject compared to a subject not treated with the Lamivudine(3TC) and the double stranded nucleic acid molecule. In one embodiment,the siNA molecule or double stranded nucleic acid molecule of theinvention is formulated as a composition described in U.S. Provisionalpatent application No. 60/678,531 and in related U.S. Provisional patentapplication No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisionalpatent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese etal.).

In one embodiment, the invention features a method for treating orpreventing Hepatitis B Virus (HBV) infection in a subject, comprisingadministering to the subject Adefovir Dipivoxil and Lamivudine (3TC) incombination with a chemically synthesized double stranded nucleic acidmolecule; wherein (a) the double stranded nucleic acid moleculecomprises a sense strand and an antisense strand; (b) each strand of thedouble stranded nucleic acid molecule is 15 to 28 nucleotides in length;(c) at least 15 nucleotides of the sense strand are complementary to theantisense strand (d) the antisense strand of the double stranded nucleicacid molecule has complementarity to a Hepatitis B Virus (HBV) targetRNA; (e) at least 20% of the internal nucleotides of each strand of thedouble stranded nucleic acid molecule are modified nucleosides having asugar modification; and (f) at least two of the sugar modifications aredifferent from each other, and wherein the Adefovir Dipivoxil andLamivudine (3TC) and the double stranded nucleic acid molecule areadministered under conditions suitable for reducing or inhibiting thelevel of Hepatitis B Virus (HBV) in the subject compared to a subjectnot treated with the Adefovir Dipivoxil and Lamivudine (3TC) and thedouble stranded nucleic acid molecule. In one embodiment, the siNAmolecule or double stranded nucleic acid molecule of the invention isformulated as a composition described in U.S. Provisional patentapplication No. 60/678,531 and in related U.S. Provisional patentapplication No. 60/703,946, filed Jul. 29, 2005, and U.S. Provisionalpatent application No. 60/737,024, filed Nov. 15, 2005 (Vargeese etal.).

In one embodiment, the invention features a composition comprisingAdefovir Dipivoxil and one or more double stranded nucleic acidmolecules or siNA molecules of the invention in a pharmaceuticallyacceptable carrier or diluent. In another embodiment, the inventionfeatures a composition comprising Adefovir Dipivoxil, Lamivudine, andone or more double stranded nucleic acid molecules or siNA molecules ofthe invention in a pharmaceutically acceptable carrier or diluent.

In one embodiment, the invention features a method for treating orpreventing an age-related disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the age-related disease, disorder, trait orcondition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the age-related disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the age-related disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of age-related diseases, traits, disorders,or conditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing a neurologic or neurodegenerative disease, disorder, trait orcondition in a subject or organism comprising contacting the subject ororganism with a siNA molecule of the invention under conditions suitableto modulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the neurologic orneurodegenerative disease, disorder, trait or condition can be achieved.In one embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via local administrationto relevant tissues or cells, such as cells and tissues involved in theneurologic or neurodegenerative disease, disorder, trait or condition.In one embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the neurologic or neurodegenerativedisease, disorder, trait or condition in a subject or organism. The siNAmolecule of the invention can be formulated or conjugated as describedherein or otherwise known in the art to target appropriate tissues orcells in the subject or organism. The siNA molecule can be combined withother therapeutic treatments and modalities as are known in the art forthe treatment of or prevention of neurologic or neurodegenerativediseases, traits, disorders, or conditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing a respiratory disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the respiratory disease, disorder, trait orcondition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the respiratory disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the respiratory disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of respiratory diseases, traits, disorders,or conditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing an ocular disease, disorder, trait or condition in a subjector organism comprising contacting the subject or organism with a siNAmolecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the ocular disease, disorder, trait orcondition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the ocular disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the ocular disease, disorder, traitor condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of ocular diseases, traits, disorders, orconditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing a dermatological disease, disorder, trait or condition in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the dermatological disease, disorder, traitor condition can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, such as cells andtissues involved in the dermatological disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the dermatological disease,disorder, trait or condition in a subject or organism. The siNA moleculeof the invention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of dermatological diseases, traits,disorders, or conditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing a liver disease, disorder; trait or condition (e.g.,hepatitis, HCV, HBV, diabetis, cirrhosis, hepatocellular carcinoma etc.)in a subject or organism comprising contacting the subject or organismwith a siNA molecule of the invention under conditions suitable tomodulate the expression of the target gene in the subject or organismwhereby the treatment or prevention of the liver disease, disorder,trait or condition can be achieved. In one embodiment, the inventionfeatures contacting the subject or organism with a siNA molecule of theinvention via local administration to relevant tissues or cells, such asliver cells and tissues involved in the liver disease, disorder, traitor condition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the liver disease, disorder, traitor condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of liver diseases, traits, disorders, orconditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing a kidney/renal disease, disorder, trait or condition (e.g.,polycystic kidney disease etc.) in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate the expression of the target genein the subject or organism whereby the treatment or prevention of thekidney/renal disease, disorder, trait or condition can be achieved. Inone embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via local administrationto relevant tissues or cells, such as kidney/renal cells and tissuesinvolved in the kidney/renal disease, disorder, trait or condition. Inone embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the kidney/renal disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of kidney diseases, traits, disorders, orconditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing an auditory disease, disorder, trait or condition (e.g.,hearing loss, deafness, etc.) in a subject or organism comprisingcontacting the subject or organism with a siNA molecule of the inventionunder conditions suitable to modulate the expression of the target genein the subject or organism whereby the treatment or prevention of theauditory disease, disorder, trait or condition can be achieved. In oneembodiment, the invention features contacting the subject or organismwith a siNA molecule of the invention via local administration torelevant tissues or cells, such as cells and tissues of the ear, innerhear, or middle ear involved in the auditory disease, disorder, trait orcondition. In one embodiment, the invention features contacting thesubject or organism with a siNA molecule of the invention via systemicadministration (such as via intravenous or subcutaneous administrationof siNA) to relevant tissues or cells, such as tissues or cells involvedin the maintenance or development of the auditory disease, disorder,trait or condition in a subject or organism. The siNA molecule of theinvention can be formulated or conjugated as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism. The siNA molecule can be combined with othertherapeutic treatments and modalities as are known in the art for thetreatment of or prevention of auditory diseases, traits, disorders, orconditions in a subject or organism.

In one embodiment, the invention features a method for treating orpreventing one or more metabolic diseases, traits, or conditions in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby thetreatment or prevention of the metabolic disease(s), trait(s), orcondition(s) can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells. In oneembodiment, the invention features contacting the subject or organismwith a siNA molecule of the invention via systemic administration (suchas via intravenous, intramuscular, subcutaneous, or GI administration ofsiNA) to relevant tissues or cells, such as tissues or cells involved inthe maintenance or development of the metabolic disease, trait, orcondition in a subject or organism (e.g., liver, pancreas, smallintestine, adipose tissue or cells). The siNA molecule of the inventioncan be formulated or conjugated as described herein or otherwise knownin the art to target appropriate tissues or cells in the subject ororganism (e.g., liver, pancreas, small intestine, adipose tissue orcells). The siNA molecule can be combined with other therapeutictreatments and modalities as are known in the art for the treatment ofor prevention of metabolic diseases, traits, or conditions in a subjector organism. In one embodiment, the metabolic disease is selected fromthe group consisting of hypercholesterolemia, hyperlipidemia,dyslipidemia, diabetis (e.g., type I and/or type II diabetis), insulinresistance, obesity, or related conditions, including but not limited tosleep apnea, hiatal hernia, reflux esophagisitis, osteoarthritis, gout,cancers associated with weight gain, gallstones, kidney stones,pulmonary hypertension, infertility, cardiovascular disease, abovenormal weight, and above normal lipid levels, uric acid levels, oroxalate levels.

In one embodiment, the invention features a method for treating orpreventing one or more metabolic diseases, traits, or conditions in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate(e.g., inhibit) the expression of an inhibitor of gene expression in thesubject or organism. In one embodiment, the inhibitor of gene expressionis a miRNA.

In one embodiment, the invention features a method for treating orpreventing one or more cardiovascular diseases, traits, or conditions ina subject or organism comprising contacting the subject or organism witha siNA molecule of the invention under conditions suitable to modulatethe expression of the target gene in the subject or organism whereby thetreatment or prevention of the cardiovascular disease(s), trait(s), orcondition(s) can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, e.g., liver,pancreas, small intestine, adipose tissue or cells tissues or cells. Inone embodiment, the invention features contacting the subject ororganism with a siNA molecule of the invention via systemicadministration (such as via intravenous, intramuscular, subcutaneous, orGI administration of siNA) to relevant tissues or cells, such as tissuesor cells involved in the maintenance or development of thecardiovascular disease, trait, or condition in a subject or organism.The siNA molecule of the invention can be formulated or conjugated asdescribed herein or otherwise known in the art to target appropriatetissues or cells in the subject or organism. The siNA molecule can becombined with other therapeutic treatments and modalities as are knownin the art for the treatment of or prevention of cardiovasculardiseases, traits, or conditions in a subject or organism. In oneembodiment the cardiovascular disease is selected from the groupconsisting of hypertension, coronary thrombosis, stroke, lipidsyndromes, hyperglycemia, hypertriglyceridemia, hyperlipidemia,ischemia, congestive heart failure, and myocardial infarction.

In one embodiment, the invention features a method for treating orpreventing one or more cardiovascular diseases, traits, or conditions ina subject or organism comprising contacting the subject or organism witha siNA molecule of the invention under conditions suitable to modulate(e.g., inhibit) the expression of an inhibitor of gene expression in thesubject or organism. In one embodiment, the inhibitor of gene expressionis a miRNA.

In one embodiment, the invention features a method for weight loss in asubject or organism comprising contacting the subject or organism with asiNA molecule of the invention under conditions suitable to modulate theexpression of the target gene in the subject or organism whereby theweight loss can be achieved. In one embodiment, the invention featurescontacting the subject or organism with a siNA molecule of the inventionvia local administration to relevant tissues or cells, e.g., liver,pancreas, small intestine, adipose tissue or cells. In one embodiment,the invention features contacting the subject or organism with a siNAmolecule of the invention via systemic administration (such as viaintravenous, intramuscular, subcutaneous, or GI administration of siNA)to relevant tissues or cells. The siNA molecule of the invention can beformulated or conjugated as described herein or otherwise known in theart to target appropriate tissues or cells in the subject or organism.The siNA molecule can be combined with other therapeutic treatments andmodalities as are known in the art for weight loss in a subject ororganism.

In one embodiment, the siNA molecule or double stranded nucleic acidmolecule of the invention is formulated as a composition described inU.S. Provisional patent application No. 60/678,531 and in related U.S.Provisional patent application No. 60/703,946, filed Jul. 29, 2005, U.S.Provisional patent application No. 60/737,024, filed Nov. 15, 2005, andU.S. Ser. No. 11/353,630, filed Feb. 14, 2006 (Vargeese et al.).

In any of the methods herein for modulating the expression of one ormore targets or for treating or preventing diseases, traits, conditions,or phenotypes in a cell, subject, or organism, the siNA molecule of theinvention modulates expression of one or more targets via RNAinterference. In one embodiment, the RNA interference is RISC mediatedcleavage of the target (e.g., siRNA mediated RNA interference). In oneembodiment, the RNA interference is translational inhibition of thetarget (e.g., miRNA mediated RNA interference). In one embodiment, theRNA interference is transcriptional inhibition of the target (e.g.,siRNA mediated transcriptional silencing). In one embodiment, the RNAinterference takes place in the cytoplasm. In one embodiment, the RNAinterference takes place in the nucleus.

In any of the methods of treatment of the invention, the siNA can beadministered to the subject as a course of treatment, for exampleadministration at various time intervals, such as once per day over thecourse of treatment, once every two days over the course of treatment,once every three days over the course of treatment, once every four daysover the course of treatment, once every five days over the course oftreatment, once every six days over the course of treatment, once perweek over the course of treatment, once every other week over the courseof treatment, once per month over the course of treatment, etc. In oneembodiment, the course of treatment is once every 1, 2, 3, 4, 5, 6, 7,8, 9, or 10 weeks. In one embodiment, the course of treatment is fromabout one to about 52 weeks or longer (e.g., indefinitely). In oneembodiment, the course of treatment is from about one to about 48 monthsor longer (e.g., indefinitely).

In one embodiment, a course of treatment involves an initial course oftreatment, such as once every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or moreweeks for a fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×,10× or more) followed by a maintenance course of treatment, such as onceevery 4, 6, 8, 10, 15, 20, 25, 30, 35, 40, or more weeks for anadditional fixed interval (e.g., 1×, 2×, 3×, 4×, 5×, 6×, 7×, 8×, 9×, 10×or more).

In any of the methods of treatment of the invention, the siNA can beadministered to the subject systemically as described herein orotherwise known in the art, either alone as a monotherapy or incombination with additional therapies described herein or as are knownin the art. Systemic administration can include, for example, pulmonary(inhalation, nebulization etc.) intravenous, subcutaneous,intramuscular, catheterization, nasopharangeal, transdermal, ororal/gastrointestinal administration as is generally known in the art.

In one embodiment, in any of the methods of treatment or prevention ofthe invention, the siNA can be administered to the subject locally or tolocal tissues as described herein or otherwise known in the art, eitheralone as a monotherapy or in combination with additional therapies asare known in the art. Local administration can include, for example,inhalation, nebulization, catheterization, implantation, directinjection, dermal/transdermal application, stenting, ear/eye drops, orportal vein administration to relevant tissues, or any other localadministration technique, method or procedure, as is generally known inthe art.

In one embodiment, the invention features a method for administeringsiNA molecules and compositions of the invention to the inner ear,comprising, contacting the siNA with inner ear cells, tissues, orstructures, under conditions suitable for the administration. In oneembodiment, the administration comprises methods and devices asdescribed in U.S. Pat. Nos. 5,421,818, 5,476,446, 5,474,529, 6,045,528,6,440,102, 6,685,697, 6,120,484; and 5,572,594; all incorporated byreference herein and the teachings of Silverstein, 1999, Ear Nose ThroatJ., 78, 595-8, 600; and Jackson and Silverstein, 2002, Otolaryngol ClinNorth Am., 35, 639-53, and adapted for use the siNA molecules of theinvention.

In another embodiment, the invention features a method of modulating theexpression of more than one target gene in a subject or organismcomprising contacting the subject or organism with one or more siNAmolecules of the invention under conditions suitable to modulate (e.g.,inhibit) the expression of the target genes in the subject or organism.

The siNA molecules of the invention can be designed to down regulate orinhibit target gene expression through RNAi targeting of a variety ofnucleic acid molecules. In one embodiment, the siNA molecules of theinvention are used to target various DNA corresponding to a target gene,for example via heterochromatic silencing or transcriptional inhibition.In one embodiment, the siNA molecules of the invention are used totarget various RNAs corresponding to a target gene, for example via RNAtarget cleavage or translational inhibition. Non-limiting examples ofsuch RNAs include messenger RNA (mRNA), non-coding RNA (ncRNA) orregulatory elements (see for example Mattick, 2005, Science, 309,1527-1528 and Claverie, 2005, Science, 309, 1529-1530) which includesmiRNA and other small RNAs, alternate RNA splice variants of targetgene(s), post-transcriptionally modified RNA of target gene(s), pre-mRNAof target gene(s), and/or RNA templates. If alternate splicing producesa family of transcripts that are distinguished by usage of appropriateexons, the instant invention can be used to inhibit gene expressionthrough the appropriate exons to specifically inhibit or to distinguishamong the functions of gene family members. For example, a protein thatcontains an alternatively spliced transmembrane domain can be expressedin both membrane bound and secreted forms. Use of the invention totarget the exon containing the transmembrane domain can be used todetermine the functional consequences of pharmaceutical targeting of themembrane bound as opposed to the secreted form of the protein.Non-limiting examples of applications of the invention relating totargeting these RNA molecules include therapeutic pharmaceuticalapplications, cosmetic applications, veterinary applications,pharmaceutical discovery applications, molecular diagnostic and genefunction applications, and gene mapping, for example using singlenucleotide polymorphism mapping with siNA molecules of the invention.Such applications can be implemented using known gene sequences or frompartial sequences available from an expressed sequence tag (EST).

In another embodiment, the siNA molecules of the invention are used totarget conserved sequences corresponding to a gene family or genefamilies such as gene families having homologous sequences. As such,siNA molecules targeting multiple gene or RNA targets can provideincreased therapeutic effect. In one embodiment, the invention featuresthe targeting (cleavage or inhibition of expression or function) of morethan one target gene sequence using a single siNA molecule, by targetingthe conserved sequences of the targeted target gene.

In one embodiment, siNA molecules can be used to characterize pathwaysof gene function in a variety of applications. For example, the presentinvention can be used to inhibit the activity of target gene(s) in apathway to determine the function of uncharacterized gene(s) in genefunction analysis, mRNA function analysis, or translational analysis.The invention can be used to determine potential target gene pathwaysinvolved in various diseases and conditions toward pharmaceuticaldevelopment. The invention can be used to understand pathways of geneexpression involved in, for example diseases, disorders, traits andconditions herein or otherwise known in the art.

In one embodiment, siNA molecule(s) and/or methods of the invention areused to down regulate the expression of gene(s) that encode RNA referredto by Genbank Accession, for example, target genes encoding RNAsequence(s) referred to herein by Genbank Accession number, for example,Genbank Accession Nos. described in U.S. Provisional Patent ApplicationNo. 60/363,124, U.S. Ser. No. 10/923,536 and PCT/US03/05028, allincorporated by reference herein.

In one embodiment, the invention features a method comprising: (a)generating a library of siNA constructs having a predeterminedcomplexity; and (b) assaying the siNA constructs of (a) above, underconditions suitable to determine RNAi target sites within the target RNAsequence. In one embodiment, the siNA molecules of (a) have strands of afixed length, for example, about 23 nucleotides in length. In anotherembodiment, the siNA molecules of (a) are of differing length, forexample having strands of about 15 to about 30 (e.g., about 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides inlength. In one embodiment, the assay can comprise a reconstituted invitro siNA assay as described herein. In another embodiment, the assaycan comprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In one embodiment, the invention features a method comprising: (a)generating a randomized library of siNA constructs having apredetermined complexity, such as of 4^(N), where N represents thenumber of base paired nucleotides in each of the siNA construct strands(e.g., for a siNA construct having 21 nucleotide sense and antisensestrands with 19 base pairs, the complexity would be 4¹⁹); and (b)assaying the siNA constructs of (a) above, under conditions suitable todetermine RNAi target sites within the target RNA sequence. In anotherembodiment, the siNA molecules of (a) have strands of a fixed length,for example about 23 nucleotides in length. In yet another embodiment,the siNA molecules of (a) are of differing length, for example havingstrands of about 15 to about 30 (e.g., about 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30) nucleotides in length. In oneembodiment, the assay can comprise a reconstituted in vitro siNA assayas described in Example 6 herein. In another embodiment, the assay cancomprise a cell culture system in which target RNA is expressed. Inanother embodiment, fragments of target RNA are analyzed for detectablelevels of cleavage, for example, by gel electrophoresis, northern blotanalysis, or RNAse protection assays, to determine the most suitabletarget site(s) within the target RNA sequence. The target RNA sequencecan be obtained as is known in the art, for example, by cloning and/ortranscription for in vitro systems, and by cellular expression in invivo systems.

In another embodiment, the invention features a method comprising: (a)analyzing the sequence of a RNA target encoded by a target gene; (b)synthesizing one or more sets of siNA molecules having sequencecomplementary to one or more regions of the RNA of (a); and (c) assayingthe siNA molecules of (b) under conditions suitable to determine RNAitargets within the target RNA sequence. In one embodiment, the siNAmolecules of (b) have strands of a fixed length, for example about 23nucleotides in length. In another embodiment, the siNA molecules of (b)are of differing length, for example having strands of about 15 to about30 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,29, or 30) nucleotides in length. In one embodiment, the assay cancomprise a reconstituted in vitro siNA assay as described herein. Inanother embodiment, the assay can comprise a cell culture system inwhich target RNA is expressed. Fragments of target RNA are analyzed fordetectable levels of cleavage, for example by gel electrophoresis,northern blot analysis, or RNAse protection assays, to determine themost suitable target site(s) within the target RNA sequence. The targetRNA sequence can be obtained as is known in the art, for example, bycloning and/or transcription for in vitro systems, and by expression inin vivo systems.

By “target site” is meant a sequence within a target RNA that is“targeted” for cleavage mediated by a siNA construct which containssequences within its antisense region that are complementary to thetarget sequence.

By “detectable level of cleavage” is meant cleavage of target RNA (andformation of cleaved product RNAs) to an extent sufficient to discerncleavage products above the background of RNAs produced by randomdegradation of the target RNA. Production of cleavage products from 1-5%of the target RNA is sufficient to detect above the background for mostmethods of detection.

In one embodiment, the invention features a composition comprising asiNA molecule of the invention, which can be chemically-modified, in apharmaceutically acceptable carrier or diluent. In another embodiment,the invention features a pharmaceutical composition comprising siNAmolecules of the invention, which can be chemically-modified, targetingone or more genes in a pharmaceutically acceptable carrier or diluent.In another embodiment, the invention features a method for diagnosing adisease, trait, or condition in a subject comprising administering tothe subject a composition of the invention under conditions suitable forthe diagnosis of the disease, trait, or condition in the subject. Inanother embodiment, the invention features a method for treating orpreventing a disease, trait, or condition, such as metabolic and/orcardiovascular diseases, trait, conditions, or disorders in a subject,comprising administering to the subject a composition of the inventionunder conditions suitable for the treatment or prevention of thedisease, trait, or condition in the subject, alone or in conjunctionwith one or more other therapeutic compounds.

In another embodiment, the invention features a method for validating atarget gene target, comprising: (a) synthesizing a siNA molecule of theinvention, which can be chemically-modified, wherein one of the siNAstrands includes a sequence complementary to RNA of a target gene; (b)introducing the siNA molecule into a cell, tissue, subject, or organismunder conditions suitable for modulating expression of the target genein the cell, tissue, subject, or organism; and (c) determining thefunction of the gene by assaying for any phenotypic change in the cell,tissue, subject, or organism.

In another embodiment, the invention features a method for validating atarget comprising: (a) synthesizing a siNA molecule of the invention,which can be chemically-modified, wherein one of the siNA strandsincludes a sequence complementary to RNA of a target gene; (b)introducing the siNA molecule into a biological system under conditionssuitable for modulating expression of the target gene in the biologicalsystem; and (c) determining the function of the gene by assaying for anyphenotypic change in the biological system.

By “biological system” is meant, material, in a purified or unpurifiedform, from biological sources, including but not limited to human oranimal, wherein the system comprises the components required for RNAiactivity. The term “biological system” includes, for example, a cell,tissue, subject, or organism, or extract thereof. The term biologicalsystem also includes reconstituted RNAi systems that can be used in anin vitro setting.

By “phenotypic change” is meant any detectable change to a cell thatoccurs in response to contact or treatment with a nucleic acid moleculeof the invention (e.g., siNA). Such detectable changes include, but arenot limited to, changes in shape, size, proliferation, motility, proteinexpression or RNA expression or other physical or chemical changes ascan be assayed by methods known in the art. The detectable change canalso include expression of reporter genes/molecules such as GreenFlorescent Protein (GFP) or various tags that are used to identify anexpressed protein or any other cellular component that can be assayed.

In one embodiment, the invention features a kit containing a siNAmolecule of the invention, which can be chemically-modified, that can beused to modulate the expression of a target gene in a biological system,including, for example, in a cell, tissue, subject, or organism. Inanother embodiment, the invention features a kit containing more thanone siNA molecule of the invention, which can be chemically-modified,that can be used to modulate the expression of more than one target genein a biological system, including, for example, in a cell, tissue,subject, or organism.

In one embodiment, the invention features a cell containing one or moresiNA molecules of the invention, which can be chemically-modified. Inanother embodiment, the cell containing a siNA molecule of the inventionis a mammalian cell. In yet another embodiment, the cell containing asiNA molecule of the invention is a human cell.

In one embodiment, the synthesis of a siNA molecule of the invention,which can be chemically-modified, comprises: (a) synthesis of twocomplementary strands of the siNA molecule; (b) annealing the twocomplementary strands together under conditions suitable to obtain adouble-stranded siNA molecule. In another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phaseoligonucleotide synthesis. In yet another embodiment, synthesis of thetwo complementary strands of the siNA molecule is by solid phase tandemoligonucleotide synthesis.

In one embodiment, the invention features a method for synthesizing asiNA duplex molecule comprising: (a) synthesizing a firstoligonucleotide sequence strand of the siNA molecule, wherein the firstoligonucleotide sequence strand comprises a cleavable linker moleculethat can be used as a scaffold for the synthesis of the secondoligonucleotide sequence strand of the siNA; (b) synthesizing the secondoligonucleotide sequence strand of siNA on the scaffold of the firstoligonucleotide sequence strand, wherein the second oligonucleotidesequence strand further comprises a chemical moiety than can be used topurify the siNA duplex; (c) cleaving the linker molecule of (a) underconditions suitable for the two siNA oligonucleotide strands tohybridize and form a stable duplex; and (d) purifying the siNA duplexutilizing the chemical moiety of the second oligonucleotide sequencestrand. In one embodiment, cleavage of the linker molecule in (c) abovetakes place during deprotection of the oligonucleotide, for example,under hydrolysis conditions using an alkylamine base such asmethylamine. In one embodiment, the method of synthesis comprises solidphase synthesis on a solid support such as controlled pore glass (CPG)or polystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity as thesolid support derivatized linker, such that cleavage of the solidsupport derivatized linker and the cleavable linker of (a) takes placeconcomitantly. In another embodiment, the chemical moiety of (b) thatcan be used to isolate the attached oligonucleotide sequence comprises atrityl group, for example a dimethoxytrityl group, which can be employedin a trityl-on synthesis strategy as described herein. In yet anotherembodiment, the chemical moiety, such as a dimethoxytrityl group, isremoved during purification, for example, using acidic conditions.

In a further embodiment, the method for siNA synthesis is a solutionphase synthesis or hybrid phase synthesis wherein both strands of thesiNA duplex are synthesized in tandem using a cleavable linker attachedto the first sequence which acts a scaffold for synthesis of the secondsequence. Cleavage of the linker under conditions suitable forhybridization of the separate siNA sequence strands results in formationof the double-stranded siNA molecule.

In another embodiment, the invention features a method for synthesizinga siNA duplex molecule comprising: (a) synthesizing one oligonucleotidesequence strand of the siNA molecule, wherein the sequence comprises acleavable linker molecule that can be used as a scaffold for thesynthesis of another oligonucleotide sequence; (b) synthesizing a secondoligonucleotide sequence having complementarity to the first sequencestrand on the scaffold of (a), wherein the second sequence comprises theother strand of the double-stranded siNA molecule and wherein the secondsequence further comprises a chemical moiety than can be used to isolatethe attached oligonucleotide sequence; (c) purifying the product of (b)utilizing the chemical moiety of the second oligonucleotide sequencestrand under conditions suitable for isolating the full-length sequencecomprising both siNA oligonucleotide strands connected by the cleavablelinker and under conditions suitable for the two siNA oligonucleotidestrands, to hybridize and form a stable duplex. In one embodiment,cleavage of the linker molecule in (c) above takes place duringdeprotection of the oligonucleotide, for example, under hydrolysisconditions. In another embodiment, cleavage of the linker molecule in(c) above takes place after deprotection of the oligonucleotide. Inanother embodiment, the method of synthesis comprises solid phasesynthesis on a solid support such as controlled pore glass (CPG) orpolystyrene, wherein the first sequence of (a) is synthesized on acleavable linker, such as a succinyl linker, using the solid support asa scaffold. The cleavable linker in (a) used as a scaffold forsynthesizing the second strand can comprise similar reactivity ordiffering reactivity as the solid support derivatized linker, such thatcleavage of the solid support derivatized linker and the cleavablelinker of (a) takes place either concomitantly or sequentially. In oneembodiment, the chemical moiety of (b) that can be used to isolate theattached oligonucleotide sequence comprises a trityl group, for examplea dimethoxytrityl group.

In another embodiment, the invention features a method for making adouble-stranded siNA molecule in a single synthetic process comprising:(a) synthesizing an oligonucleotide having a first and a secondsequence, wherein the first sequence is complementary to the secondsequence, and the first oligonucleotide sequence is linked to the secondsequence via a cleavable linker, and wherein a terminal 5′-protectinggroup, for example, a 5′-O-dimethoxytrityl group (5′-O-DMT) remains onthe oligonucleotide having the second sequence; (b) deprotecting theoligonucleotide whereby the deprotection results in the cleavage of thelinker joining the two oligonucleotide sequences; and (c) purifying theproduct of (b) under conditions suitable for isolating thedouble-stranded siNA molecule, for example using a trityl-on synthesisstrategy as described herein.

In another embodiment, the method of synthesis of siNA molecules of theinvention comprises the teachings of Scaringe et al., U.S. Pat. Nos.5,889,136; 6,008,400; and 6,111,086, incorporated by reference herein intheir entirety.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide (e.g., RNA or DNA target), whereinthe siNA construct comprises one or more chemical modifications, forexample, one or more chemical modifications having any of Formulae I-VIIor any combination thereof that increases the nuclease resistance of thesiNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased nuclease resistance comprising (a)introducing nucleotides having any of Formula I-VII or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules havingincreased nuclease resistance.

In another embodiment, the invention features a method for generatingsiNA molecules with improved toxicologic profiles (e.g., havingattenuated or no immunostimulatory properties) comprising (a)introducing nucleotides having any of Formula I-VII (e.g., siNA motifsreferred to in Table I) or any combination thereof into a siNA molecule,and (b) assaying the siNA molecule of step (a) under conditions suitablefor isolating siNA molecules having improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA formulations with improved toxicologic profiles (e.g., havingattenuated or no immunostimulatory properties) comprising (a) generatinga siNA formulation comprising a siNA molecule of the invention and adelivery vehicle or delivery particle as described herein or asotherwise known in the art, and (b) assaying the siNA formulation ofstep (a) under conditions suitable for isolating siNA formulationshaving improved toxicologic profiles.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) introducing nucleotides having anyof Formula I-VII (e.g., siNA motifs referred to in Table I) or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules that do not stimulate an interferon response.

In another embodiment, the invention features a method for generatingsiNA formulations that do not stimulate an interferon response (e.g., nointerferon response or attenuated interferon response) in a cell,subject, or organism, comprising (a) generating a siNA formulationcomprising a siNA molecule of the invention and a delivery vehicle ordelivery particle as described herein or as otherwise known in the art,and (b) assaying the siNA formulation of step (a) under conditionssuitable for isolating siNA formulations that do not stimulate aninterferon response. In one embodiment, the interferon comprisesinterferon alpha.

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate an inflammatory or proinflammatorycytokine response (e.g., no cytokine response or attenuated cytokineresponse) in a cell, subject, or organism, comprising (a) introducingnucleotides having any of Formula I-VII (e.g., siNA motifs referred toin Table I) or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules that do not stimulate a cytokine response. Inone embodiment, the cytokine comprises an interleukin such asinterleukin-6 (IL-6) and/or tumor necrosis alpha (TNF-α).

In another embodiment, the invention features a method for generatingsiNA formulations that do not stimulate an inflammatory orproinflammatory cytokine response (e.g., no cytokine response orattenuated cytokine response) in a cell, subject, or organism,comprising (a) generating a siNA formulation comprising a siNA moleculeof the invention and a delivery vehicle or delivery particle asdescribed herein or as otherwise known in the art, and (b) assaying thesiNA formulation of step (a) under conditions suitable for isolatingsiNA formulations that do not stimulate a cytokine response. In oneembodiment, the cytokine comprises an interleukin such as interleukin-6(IL-6) and/or tumor necrosis alpha (TNF-α).

In another embodiment, the invention features a method for generatingsiNA molecules that do not stimulate Toll-like Receptor (TLR) response(e.g., no TLR response or attenuated TLR response) in a cell, subject,or organism, comprising (a) introducing nucleotides having any ofFormula (e.g., siNA motifs referred to in Table I) or any combinationthereof into a siNA molecule, and (b) assaying the siNA molecule of step(a) under conditions suitable for isolating siNA molecules that do notstimulate a TLR response. In one embodiment, the TLR comprises TLR3,TLR7, TLR8 and/or TLR9.

In another embodiment, the invention features a method for generatingsiNA formulations that do not stimulate a Toll-like Receptor (TLR)response (e.g., no TLR response or attenuated TLR response) in a cell,subject, or organism, comprising (a) generating a siNA formulationcomprising a siNA molecule of the invention and a delivery vehicle ordelivery particle as described herein or as otherwise known in the art,and (b) assaying the siNA formulation of step (a) under conditionssuitable for isolating siNA formulations that do not stimulate a TLRresponse. In one embodiment, the TLR comprises TLR3, TLR7, TLR8 and/orTLR9.

In one embodiment, the invention features a chemically synthesizeddouble stranded short interfering nucleic acid (siNA) molecule thatdirects cleavage of a target RNA via RNA interference (RNAi), wherein:(a) each strand of said siNA molecule is about 18 to about 38nucleotides in length; (b) one strand of said siNA molecule comprisesnucleotide sequence having sufficient complementarity to said target RNAfor the siNA molecule to direct cleavage of the target RNA via RNAinterference; and (c) wherein the nucleotide positions within said siNAmolecule are chemically modified to reduce the immunostimulatoryproperties of the siNA molecule to a level below that of a correspondingunmodified siRNA molecule. Such siNA molecules are said to have animproved toxicologic profile compared to an unmodified or minimallymodified siNA.

By “improved toxicologic profile”, is meant that the chemically modifiedor formulated siNA construct exhibits decreased toxicity in a cell,subject, or organism compared to an unmodified or unformulated siNA, orsiNA molecule having fewer modifications or modifications that are lesseffective in imparting improved toxicology. Such siNA molecules are alsoconsidered to have “improved RNAi activity”. In a non-limiting example,siNA molecules and formulations with improved toxicologic profiles areassociated with reduced immunostimulatory properties, such as a reduced,decreased or attenuated immunostimulatory response in a cell, subject,or organism compared to an unmodified or unformulated siNA, or siNAmolecule having fewer modifications or modifications that are lesseffective in imparting improved toxicology. Such an improved toxicologicprofile is characterized by abrogated or reduced immunostimulation, suchas reduction or abrogation of induction of interferons (e.g., interferonalpha), inflammatory cytokines (e.g., interleukins such as IL-6, and/orTNF-alpha), and/or toll like receptors (e.g., TLR-3, TLR-7, TLR-8,and/or TLR-9). In one embodiment, a siNA molecule or formulation with animproved toxicological profile comprises no ribonucleotides. In oneembodiment, a siNA molecule or formulation with an improvedtoxicological profile comprises less than 5 ribonucleotides (e.g., 1, 2,3, or 4 ribonucleotides). In one embodiment, a siNA molecule orformulation with an improved toxicological profile comprises Stab 7,Stab 8, Stab 11, Stab 12, Stab 13, Stab 16, Stab 17, Stab 18, Stab 19,Stab 20, Stab 23, Stab 24, Stab 25, Stab 26, Stab 27, Stab 28, Stab 29,Stab 30, Stab 31, Stab 32, Stab 33, Stab 34, Stab 35, Stab 36 or anycombination thereof (see Table I). Herein, numeric Stab chemistriesinclude both 2′-fluoro and 2′-OCF3 versions of the chemistries shown inTable I. For example, “Stab 7/8” refers to both Stab 7/8 and Stab 7F/8Fetc. In one embodiment, a siNA molecule or formulation with an improvedtoxicological profile comprises a siNA molecule of the invention and aformulation as described in United States Patent Application PublicationNo. 20030077829, incorporated by reference herein in its entiretyincluding the drawings.

In one embodiment, the level of immunostimulatory response associatedwith a given siNA molecule can be measured as is described herein or asis otherwise known in the art, for example by determining the level ofPKR/interferon response, proliferation, B-cell activation, and/orcytokine production in assays to quantitate the immunostimulatoryresponse of particular siNA molecules (see, for example, Leifer et al.,2003, J Immnunother. 26, 313-9; and U.S. Pat. No. 5,968,909,incorporated in its entirety by reference). In one embodiment, thereduced immunostimulatory response is between about 10% and about 100%compared to an unmodified or minimally modified siRNA molecule, e.g.,about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% reducedimmunostimulatory response. In one embodiment, the immunostimulatoryresponse associated with a siNA molecule can be modulated by the degreeof chemical modification. For example, a siNA molecule having betweenabout 10% and about 100%, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% or at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% or 100% of the nucleotide positions in the siNA moleculemodified can be selected to have a corresponding degree ofimmunostimulatory properties as described herein.

In one embodiment, the degree of reduced immunostimulatory response isselected for optimized RNAi activity. For example, retaining a certaindegree of immunostimulation can be preferred to treat viral infection,where less than 100% reduction in immunostimulation may be preferred formaximal antiviral activity (e.g., about 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, or 90% reduction in immunostimulation) whereas the inhibitionof expression of an endogenous gene target may be preferred with siNAmolecules that possess minimal immunostimulatory properties to preventnon-specific toxicity or off target effects (e.g., about 90% to about100% reduction in immunostimulation).

In one embodiment, the invention features a chemically synthesizeddouble stranded siNA molecule that directs cleavage of a target RNA viaRNA interference (RNAi), wherein (a) each strand of said siNA moleculeis about 18 to about 38 nucleotides in length; (b) one strand of saidsiNA molecule comprises nucleotide sequence having sufficientcomplementarity to said target RNA for the siNA molecule to directcleavage of the target RNA via RNA interference; and (c) wherein one ormore nucleotides of said siNA molecule are chemically modified to reducethe immunostimulatory properties of the siNA molecule to a level belowthat of a corresponding unmodified siNA molecule. In one embodiment,each strand comprises at least about 18 nucleotides that arecomplementary to the nucleotides of the other strand.

In another embodiment, the siNA molecule comprising modified nucleotidesto reduce the immunostimulatory properties of the siNA moleculecomprises an antisense region having nucleotide sequence that iscomplementary to a nucleotide sequence of a target gene or a portionthereof and further comprises a sense region, wherein said sense regioncomprises a nucleotide sequence substantially similar to the nucleotidesequence of said target gene or portion thereof. In one embodimentthereof, the antisense region and the sense region comprise about 18 toabout 38 nucleotides, wherein said antisense region comprises at leastabout 18 nucleotides that are complementary to nucleotides of the senseregion. In one embodiment thereof, the pyrimidine nucleotides in thesense region are 2′-O-methyl pyrimidine nucleotides. In anotherembodiment thereof, the purine nucleotides in the sense region are2′-deoxy purine nucleotides. In yet another embodiment thereof, thepyrimidine nucleotides present in the sense region are2′-deoxy-2′-fluoro pyrimidine nucleotides. In another embodimentthereof, the pyrimidine nucleotides of said antisense region are2′-deoxy-2′-fluoro pyrimidine nucleotides. In yet another embodimentthereof, the purine nucleotides of said antisense region are 2′-O-methylpurine nucleotides. In still another embodiment thereof, the purinenucleotides present in said antisense region comprise 2′-deoxypurinenucleotides. In another embodiment, the antisense region comprises aphosphorothioate internucleotide linkage at the 3′ end of said antisenseregion. In another embodiment, the antisense region comprises a glycerylmodification at a 3′ end of said antisense region.

In other embodiments, the siNA molecule comprising modified nucleotidesto reduce the immunostimulatory properties of the siNA molecule cancomprise any of the structural features of siNA molecules describedherein. In other embodiments, the siNA molecule comprising modifiednucleotides to reduce the immunostimulatory properties of the siNAmolecule can comprise any of the chemical modifications of siNAmolecules described herein.

In one embodiment, the invention features a method for generating achemically synthesized double stranded siNA molecule having chemicallymodified nucleotides to reduce the immunostimulatory properties of thesiNA molecule, comprising (a) introducing one or more modifiednucleotides in the siNA molecule, and (b) assaying the siNA molecule ofstep (a) under conditions suitable for isolating an siNA molecule havingreduced immunostimulatory properties compared to a corresponding siNAmolecule having unmodified nucleotides. Each strand of the siNA moleculeis about 18 to about 38 nucleotides in length. One strand of the siNAmolecule comprises nucleotide sequence having sufficient complementarityto the target RNA for the siNA molecule to direct cleavage of the targetRNA via RNA interference. In one embodiment, the reducedimmunostimulatory properties comprise an abrogated or reduced inductionof inflammatory or proinflammatory cytokines, such as interleukin-6(IL-6) or tumor necrosis alpha (TNF-α), in response to the siNA beingintroduced in a cell, tissue, or organism. In another embodiment, thereduced immunostimulatory properties comprise an abrogated or reducedinduction of Toll Like Receptors (TLRs), such as TLR3, TLR7, TLR8 orTLR9, in response to the siNA being introduced in a cell, tissue, ororganism. In another embodiment, the reduced immunostimulatoryproperties comprise an abrogated or reduced induction of interferons,such as interferon alpha, in response to the siNA being introduced in acell, tissue, or organism.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the sense and antisense strandsof the siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the sense andantisense strands of the siNA molecule comprising (a) introducingnucleotides having any of Formula or any combination thereof into a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having increasedbinding affinity between the sense and antisense strands of the siNAmolecule.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary target RNA sequence within a cell.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the binding affinity between the antisense strand of the siNAconstruct and a complementary target DNA sequence within a cell.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target RNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target RNA sequence.

In another embodiment, the invention features a method for generatingsiNA molecules with increased binding affinity between the antisensestrand of the siNA molecule and a complementary target DNA sequencecomprising (a) introducing nucleotides having any of Formula I-VII orany combination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having increased binding affinity between the antisense strandof the siNA molecule and a complementary target DNA sequence.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulate the polymerase activity of a cellular polymerase capable ofgenerating additional endogenous siNA molecules having sequence homologyto the chemically-modified siNA construct.

In another embodiment, the invention features a method for generatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to a chemically-modified siNAmolecule comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules capable of mediating increased polymerase activity of acellular polymerase capable of generating additional endogenous siNAmolecules having sequence homology to the chemically-modified siNAmolecule.

In one embodiment, the invention features chemically-modified siNAconstructs that mediate RNAi against a target polynucleotide in a cell,wherein the chemical modifications do not significantly effect theinteraction of siNA with a target RNA molecule, DNA molecule and/orproteins or other factors that are essential for RNAi in a manner thatwould decrease the efficacy of RNAi mediated by such siNA constructs.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi specificity against polynucleotidetargets comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved RNAi specificity. In one embodiment,improved specificity comprises having reduced off target effectscompared to an unmodified siNA molecule. For example, introduction ofterminal cap moieties at the 3′-end, 5′-end, or both 3′ and 5′-ends ofthe sense strand or region of a siNA molecule of the invention candirect the siNA to have improved specificity by preventing the sensestrand or sense region from acting as a template for RNAi activityagainst a corresponding target having complementarity to the sensestrand or sense region.

In another embodiment, the invention features a method for generatingsiNA molecules with improved RNAi activity against a targetpolynucleotide comprising (a) introducing nucleotides having any ofFormula I-VII or any combination thereof into a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved RNAi activity.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a targetRNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target RNA.

In yet another embodiment, the invention features a method forgenerating siNA molecules with improved RNAi activity against a targetDNA comprising (a) introducing nucleotides having any of Formula I-VIIor any combination thereof into a siNA molecule, and (b) assaying thesiNA molecule of step (a) under conditions suitable for isolating siNAmolecules having improved RNAi activity against the target DNA.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatmodulates the cellular uptake of the siNA construct, such as cholesterolconjugation of the siNA.

In another embodiment, the invention features a method for generatingsiNA molecules against a target polynucleotide with improved cellularuptake comprising (a) introducing nucleotides having any of FormulaI-VII or any combination thereof into a siNA molecule, and (b) assayingthe siNA molecule of step (a) under conditions suitable for isolatingsiNA molecules having improved cellular uptake.

In one embodiment, the invention features siNA constructs that mediateRNAi against a target polynucleotide, wherein the siNA constructcomprises one or more chemical modifications described herein thatincreases the bioavailability of the siNA construct, for example, byattaching polymeric conjugates such as polyethyleneglycol or equivalentconjugates that improve the pharmacokinetics of the siNA construct, orby attaching conjugates that target specific tissue types or cell typesin vivo. Non-limiting examples of such conjugates are described inVargeese et al., U.S. Ser. No. 10/201,394 incorporated by referenceherein.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved bioavailability comprising (a)introducing a conjugate into the structure of a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchconjugates can include ligands for cellular receptors, such as peptidesderived from naturally occurring protein ligands; protein localizationsequences, including cellular ‘ZIP code sequences; antibodies; nucleicacid aptamers; vitamins and other co-factors, such as folate andN-acetylgalactosamine; polymers, such as polyethyleneglycol (PEG);phospholipids; cholesterol; cholesterol derivatives, polyamines, such asspermine or spermidine; and others.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence is chemically modified in amanner that it can no longer act as a guide sequence for efficientlymediating RNA interference and/or be recognized by cellular proteinsthat facilitate RNAi. In one embodiment, the first nucleotide sequenceof the siNA is chemically modified as described herein. In oneembodiment, the first nucleotide sequence of the siNA is not modified(e.g., is all RNA).

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein the second sequence is designed or modified in amanner that prevents its entry into the RNAi pathway as a guide sequenceor as a sequence that is complementary to a target nucleic acid (e.g.,RNA) sequence. In one embodiment, the first nucleotide sequence of thesiNA is chemically modified as described herein. In one embodiment, thefirst nucleotide sequence of the siNA is not modified (e.g., is allRNA). Such design or modifications are expected to enhance the activityof siNA and/or improve the specificity of siNA molecules of theinvention. These modifications are also expected to minimize anyoff-target effects and/or associated toxicity.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarily to said firstsequence, wherein said second sequence is incapable of acting as a guidesequence for mediating RNA interference. In one embodiment, the firstnucleotide sequence of the siNA is chemically modified as describedherein. In one embodiment, the first nucleotide sequence of the siNA isnot modified (e.g., is all RNA).

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence does not have a terminal5′-hydroxyl (5′-OH) or 5′-phosphate group.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarily to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end of said second sequence. In one embodiment, the terminalcap moiety comprises an inverted abasic, inverted deoxy abasic, invertednucleotide moiety, a group shown in FIG. 10, an alkyl or cycloalkylgroup, a heterocycle, or any other group that prevents RNAi activity inwhich the second sequence serves as a guide sequence or template forRNAi.

In one embodiment, the invention features a double stranded shortinterfering nucleic acid (siNA) molecule that comprises a firstnucleotide sequence complementary to a target RNA sequence or a portionthereof, and a second sequence having complementarity to said firstsequence, wherein said second sequence comprises a terminal cap moietyat the 5′-end and 3′-end of said second sequence. In one embodiment,each terminal cap moiety individually comprises an inverted abasic,inverted deoxy abasic, inverted nucleotide moiety, a group shown in FIG.10, an alkyl or cycloalkyl group, a heterocycle, or any other group thatprevents RNAi activity in which the second sequence serves as a guidesequence or template for RNAi.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising (a) introducingone or more chemical modifications into the structure of a siNAmolecule, and (b) assaying the siNA molecule of step (a) underconditions suitable for isolating siNA molecules having improvedspecificity. In another embodiment, the chemical modification used toimprove specificity comprises terminal cap modifications at the 5′-end,3′-end, or both 5′ and 3′-ends of the siNA molecule. The terminal capmodifications can comprise, for example, structures shown in FIG. 10(e.g., inverted deoxyabasic moieties) or any other chemical modificationthat renders a portion of the siNA molecule (e.g., the sense strand)incapable of mediating RNA interference against an off target nucleicacid sequence. In a non-limiting example, a siNA molecule is designedsuch that only the antisense sequence of the siNA molecule can serve asa guide sequence for RISC mediated degradation of a corresponding targetRNA sequence. This can be accomplished by rendering the sense sequenceof the siNA inactive by introducing chemical modifications to the sensestrand that preclude recognition of the sense strand as a guide sequenceby RNAi machinery. In one embodiment, such chemical modificationscomprise any chemical group at the 5′-end of the sense strand of thesiNA, or any other group that serves to render the sense strand inactiveas a guide sequence for mediating RNA interference. These modifications,for example, can result in a molecule where the 5′-end of the sensestrand no longer has a free 5′-hydroxyl (5′-OH) or a free 5′-phosphategroup (e.g., phosphate, diphosphate, triphosphate, cyclic phosphateetc.). Non-limiting examples of such siNA constructs are describedherein, such as “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”,“Stab 23/24”, “Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab7, 9, 17, 23, or 24 sense strands) chemistries and variants thereof (seeTable I) wherein the 5′-end and 3′-end of the sense-strand of the siNAdo not comprise a hydroxyl group or phosphate group. Herein, numericStab chemistries include both 2′-fluoro and 2′-OCF3 versions of thechemistries shown in Table I. For example, “Stab 7/8” refers to bothStab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for generating siNAmolecules of the invention with improved specificity for down regulatingor inhibiting the expression of a target nucleic acid (e.g., a DNA orRNA such as a gene or its corresponding RNA), comprising introducing oneor more chemical modifications into the structure of a siNA moleculethat prevent a strand or portion of the siNA molecule from acting as atemplate or guide sequence for RNAi activity. In one embodiment, theinactive strand or sense region of the siNA molecule is the sense strandor sense region of the siNA molecule, i.e. the strand or region of thesiNA that does not have complementarity to the target nucleic acidsequence. In one embodiment, such chemical modifications comprise anychemical group at the 5′-end of the sense strand or region of the siNAthat does not comprise a 5′-hydroxyl (5′-OH) or 5′-phosphate group, orany other group that serves to render the sense strand or sense regioninactive as a guide sequence for mediating RNA interference.Non-limiting examples of such siNA constructs are described herein, suchas “Stab 9/10”, “Stab 7/8”, “Stab 7/19”, “Stab 17/22”, “Stab 23/24”,“Stab 24/25”, and “Stab 24/26” (e.g., any siNA having Stab 7, 9, 17, 23,or 24 sense strands) chemistries and variants thereof (see Table I)wherein the 5′-end and 3′-end of the sense strand of the siNA do notcomprise a hydroxyl group or phosphate group. Herein, numeric Stabchemistries include both 2′-fluoro and 2′-OCF3 versions of thechemistries shown in Table I. For example, “Stab 7/8” refers to bothStab 7/8 and Stab 7F/8F etc.

In one embodiment, the invention features a method for screening siNAmolecules that are active in mediating RNA interference against a targetnucleic acid sequence comprising (a) generating a plurality ofunmodified siNA molecules, (b) screening the siNA molecules of step (a)under conditions suitable for isolating siNA molecules that are activein mediating RNA interference against the target nucleic acid sequence,and (c) introducing chemical modifications (e.g., chemical modificationsas described herein or as otherwise known in the art) into the activesiNA molecules of (b). In one embodiment, the method further comprisesre-screening the chemically modified siNA molecules of step (c) underconditions suitable for isolating chemically modified siNA moleculesthat are active in mediating RNA interference against the target nucleicacid sequence.

In one embodiment, the invention features a method for screeningchemically modified siNA molecules that are active in mediating RNAinterference against a target nucleic acid sequence comprising (a)generating a plurality of chemically modified siNA molecules (e.g., siNAmolecules as described herein or as otherwise known in the art), and (b)screening the siNA molecules of step (a) under conditions suitable forisolating chemically modified siNA molecules that are active inmediating RNA interference against the target nucleic acid sequence.

The term “ligand” refers to any compound or molecule, such as a drug,peptide, hormone, or neurotransmitter, that is capable of interactingwith another compound, such as a receptor, either directly orindirectly. The receptor that interacts with a ligand can be present onthe surface of a cell or can alternately be an intercellular receptor.Interaction of the ligand with the receptor can result in a biochemicalreaction, or can simply be a physical interaction or association.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing an excipient formulation to a siNA molecule, and (b)assaying the siNA molecule of step (a) under conditions suitable forisolating siNA molecules having improved bioavailability. Suchexcipients include polymers such as cyclodextrins, lipids, cationiclipids, polyamines, phospholipids, nanoparticles, receptors, ligands,and others.

In another embodiment, the invention features a method for generatingsiNA molecules of the invention with improved bioavailability comprising(a) introducing nucleotides having any of Formulae I-VII or anycombination thereof into a siNA molecule, and (b) assaying the siNAmolecule of step (a) under conditions suitable for isolating siNAmolecules having improved bioavailability.

In another embodiment, polyethylene glycol (PEG) can be covalentlyattached to siNA compounds of the present invention. The attached PEGcan be any molecular weight, preferably from about 100 to about 50,000daltons (Da).

The present invention can be used alone or as a component of a kithaving at least one of the reagents necessary to carry out the in vitroor in vivo introduction of RNA to test samples and/or subjects. Forexample, preferred components of the kit include a siNA molecule of theinvention and a vehicle that promotes introduction of the siNA intocells of interest as described herein (e.g., using lipids and othermethods of transfection known in the art, see for example Beigelman etal, U.S. Pat. No. 6,395,713). The kit can be used for target validation,such as in determining gene function and/or activity, or in drugoptimization, and in drug discovery (see for example Usman et al., U.S.Ser. No. 60/402,996). Such a kit can also include instructions to allowa user of the kit to practice the invention.

The term “short interfering nucleic acid”, “siNA”, “short interferingRNA”, “siRNA”, “short interfering nucleic acid molecule”, “shortinterfering oligonucleotide molecule”, or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expressionor viral replication by mediating RNA interference “RNAi” or genesilencing in a sequence specific manner. These terms can refer to bothindividual nucleic acid molecules, a plurality of such nucleic acidmolecules, or pools of such nucleic acid molecules. The siNA can be adouble-stranded nucleic acid molecule comprising self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense regionhaving nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof. The siNA can be assembled from twoseparate oligonucleotides, where one strand is the sense strand and theother is the antisense strand, wherein the antisense and sense strandsare self-complementary (i.e., each strand comprises nucleotide sequencethat is complementary to nucleotide sequence in the other strand; suchas where the anti sense strand and sense strand form a duplex or doublestranded structure, for example wherein the double stranded region isabout 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 to about 25 or morenucleotides of the siNA molecule are complementary to the target nucleicacid or a portion thereof). Alternatively, the siNA is assembled from asingle oligonucleotide, where the self-complementary sense and antisenseregions of the siNA are linked by means of a nucleic acid based ornon-nucleic acid-based linker(s). The siNA can be a polynucleotide witha duplex, asymmetric duplex, hairpin or asymmetric hairpin secondarystructure, having self-complementary sense and antisense regions;wherein the antisense region comprises nucleotide sequence that iscomplementary to nucleotide sequence in a separate target nucleic acidmolecule or a portion thereof and the sense region having nucleotidesequence corresponding to the target nucleic acid sequence or a portionthereof. The siNA can be a circular single-stranded polynucleotidehaving two or more loop structures and a stem comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof, and wherein the circularpolynucleotide can be processed either in vivo or in vitro to generatean active siNA molecule capable of mediating RNAi. The siNA can alsocomprise a single stranded polynucleotide having nucleotide sequencecomplementary to nucleotide sequence in a target nucleic acid moleculeor a portion thereof (for example, where such siNA molecule does notrequire the presence within the siNA molecule of nucleotide sequencecorresponding to the target nucleic acid sequence or a portion thereof),wherein the single stranded polynucleotide can further comprise aterminal phosphate group, such as a 5′-phosphate (see for exampleMartinez et al., 2002, Cell., 110, 563-574 and Schwarz et al., 2002,Molecular Cell, 10, 537-568), or 5′,3′-diphosphate. In certainembodiments, the siNA molecule of the invention comprises separate senseand antisense sequences or regions, wherein the sense and antisenseregions are covalently linked by nucleotide or non-nucleotide linkersmolecules as is known in the art, or are alternately non-covalentlylinked by ionic interactions, hydrogen bonding, van der waalsinteractions, hydrophobic interactions, and/or stacking interactions. Incertain embodiments, the siNA molecules of the invention comprisenucleotide sequence that is complementary to nucleotide sequence of atarget gene. In another embodiment, the siNA molecule of the inventioninteracts with nucleotide sequence of a target gene in a manner thatcauses inhibition of expression of the target gene. As used herein, siNAmolecules need not be limited to those molecules containing only RNA,but further encompasses chemically-modified nucleotides andnon-nucleotides. In certain embodiments, the short interfering nucleicacid molecules of the invention lack 2′-hydroxy (2′-OH) containingnucleotides. Applicant describes in certain embodiments shortinterfering nucleic acids that do not require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,short interfering nucleic acid molecules of the invention optionally donot include any ribonucleotides (e.g., nucleotides having a 2′-OHgroup). Such siNA molecules that do not require the presence ofribonucleotides within the siNA molecule to support RNAi can howeverhave an attached linker or linkers or other attached or associatedgroups, moieties, or chains containing one or more nucleotides with2′-OH groups. Optionally, siNA molecules can comprise ribonucleotides atabout 5, 10, 20, 30, 40, or 50% of the nucleotide positions. Themodified short interfering nucleic acid molecules of the invention canalso be referred to as short interfering modified oligonucleotides“siMON.” As used herein, the term siNA is meant to be equivalent toother terms used to describe nucleic acid molecules that are capable ofmediating sequence specific RNAi, for example short interfering RNA(siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpinRNA (shRNA), short interfering oligonucleotide, short interferingnucleic acid, short interfering modified oligonucleotide,chemically-modified siRNA, post-transcriptional gene silencing RNA(ptgsRNA), and others. Non limiting examples of siNA molecules of theinvention are shown in FIGS. 4-6, and Table II herein. Such siNAmolecules are distinct from other nucleic acid technologies known in theart that mediate inhibition of gene expression, such as ribozymes,antisense, triplex forming, aptamer, 2,5-A chimera, or decoyoligonucleotides.

By “RNA interference” or “RNAi” is meant a biological process ofinhibiting or down regulating gene expression in a cell as is generallyknown in the art and which is mediated by short interfering nucleic acidmolecules, see for example Zamore and Haley, 2005, Science, 309,1519-1524; Vaughn and Martienssen, 2005, Science, 309, 1525-1526; Zamoreet al., 2000, Cell, 101, 25-33; Bass, 2001, Nature, 411, 428-429;Elbashir et al., 2001, Nature, 411, 494-498; and Kreutzer et al.,International PCT Publication No. WO 00/44895; Zernicka-Goetz et al.,International PCT Publication No. WO 01/36646; Fire, International PCTPublication No. WO 99/32619; Plaetinck et al., International PCTPublication No. WO 00/01846; Mello and Fire, International PCTPublication No. WO 01/29058; Deschamps-Depaillette, International PCTPublication No. WO 99/07409; and Li et al., International PCTPublication No. WO 00/44914; Allshire, 2002, Science, 297, 1818-1819;Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science,297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237;Hutvagner and Zamore, 2002, Science, 297, 2056-60; McManus et al., 2002,RNA, 8, 842-850; Reinhart et al., 2002, gene & Dev., 16, 1616-1626; andReinhart & Bartel, 2002, Science, 297, 1831). In addition, as usedherein, the term RNAi is meant to be equivalent to other terms used todescribe sequence specific RNA interference, such as posttranscriptional gene silencing, translational inhibition,transcriptional inhibition, or epigenetics. For example, siNA moleculesof the invention can be used to epigenetically silence genes at both thepost-transcriptional level or the pre-transcriptional level. In anon-limiting example, epigenetic modulation of gene expression by siNAmolecules of the invention can result from siNA mediated modification ofchromatin structure or methylation patterns to alter gene expression(see, for example, Verdel et al., 2004, Science, 303, 672-676;Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science,297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein,2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297,2232-2237). In another non-limiting example, modulation of geneexpression by siNA molecules of the invention can result from siNAmediated cleavage of RNA (either coding or non-coding RNA) via RISC, oralternately, translational inhibition as is known in the art. In anotherembodiment, modulation of gene expression by siNA molecules of theinvention can result from transcriptional inhibition see for exampleJanowski et al., 2005, Nature Chemical Biology, 1, 216-222).

In one embodiment, a siNA molecule of the invention is a duplex formingoligonucleotide “DFO”, (see for example FIGS. 14-15 and Vaish et al.,U.S. Ser. No. 10/727,780 filed Dec. 3, 2003 and International PCTApplication No. USO4/16390, filed May 24, 2004).

In one embodiment, a siNA molecule of the invention is a multifunctionalsiNA, (see for example FIGS. 16-28 and Jadhav et al., U.S. Ser. No.60/543,480 filed Feb. 10, 2004 and International PCT Application No.USO4/16390, filed May 24, 2004). In one embodiment, the multifunctionalsiNA of the invention can comprise sequence targeting, for example, twoor more regions of target RNA (see for example target sequences inTables II and III). In one embodiment, the multifunctional siNA of theinvention can comprise sequence targeting one or more different targets,including coding regions and non-coding regions of SREBP1.

By “asymmetric hairpin” as used herein is meant a linear siNA moleculecomprising an antisense region, a loop portion that can comprisenucleotides or non-nucleotides, and a sense region that comprises fewernucleotides than the antisense region to the extent that the senseregion has enough complementary nucleotides to base pair with theantisense region and form a duplex with loop. For example, an asymmetrichairpin siNA molecule of the invention can comprise an antisense regionhaving length sufficient to mediate RNAi in a cell or in vitro system(e.g., about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and a loop regioncomprising about 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or12) nucleotides, and a sense region having about 3 to about 25 (e.g.,about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, or 25) nucleotides that are complementary to theantisense region. The asymmetric hairpin siNA molecule can also comprisea 5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule car comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example an asymmetric duplex siNA molecule of the invention cancomprise an antisense region having length sufficient to mediate RNAi ina cell or in vitro system (e.g., about 15 to about 30, or about 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides)and a sense region having about 3 to about 25 (e.g., about 0.3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or25) nucleotides that are complementary to the antisense region.

By “RNAi inhibitor” is meant any molecule that can down regulate, reduceor inhibit RNA interference function or activity in a cell or organism.An RNAi inhibitor can down regulate, reduce or inhibit RNAi (e.g., RNAimediated cleavage of a target polynucleotide, translational inhibition,or transcriptional silencing) by interaction with or interfering thefunction of any component of the RNAi pathway, including proteincomponents such as RISC, or nucleic acid components such as miRNAs orsiRNAs. A RNAi inhibitor can be a siNA molecule, an antisense molecule,an aptamer, or a small molecule that interacts with or interferes withthe function of RISC, a miRNA, or a siRNA or any other component of theRNAi pathway in a cell or organism. By inhibiting RNAi (e.g., RNAimediated cleavage of a target polynucleotide, translational inhibition,or transcriptional silencing), a RNAi inhibitor of the invention can beused to modulate (e.g, up-regulate or down regulate) the expression of atarget gene. In one embodiment, a RNA inhibitor of the invention is usedto up-regulate gene expression by interfering with (e.g., reducing orpreventing) endogenous down-regulation or inhibition of gene expressionthrough translational inhibition, transcriptional silencing, or RISCmediated cleavage of a polynucleotide (e.g., mRNA). By interfering withmechanisms of endogenous repression, silencing, or inhibition of geneexpression, RNAi inhibitors of the invention can therefore be used toup-regulate gene expression for the treatment of diseases, traits, orconditions resulting from a loss of function. In one embodiment, theterm “RNAi inhibitor” is used in place of the term “siNA” in the variousembodiments herein, for example, with the effect of increasing geneexpression for the treatment of loss of function diseases, traits,and/or conditions.

By “aptamer” or “nucleic acid aptamer” as used herein is meant apolynucleotide that binds specifically to a target molecule wherein thenucleic acid molecule has sequence that is distinct from sequencerecognized by the target molecule in its natural setting. Alternately,an aptamer can be a nucleic acid molecule that binds to a targetmolecule where the target molecule does not naturally bind to a nucleicacid. The target molecule can be any molecule of interest. For example,the aptamer can be used to bind to a ligand-binding domain of a protein,thereby preventing interaction of the naturally occurring ligand withthe protein. This is a non-limiting example and those in the art willrecognize that other embodiments can be readily generated usingtechniques generally known in the art, see for example Gold et al.,1995, Annu. Rev. Biochem., 64, 763; Brody and Gold, 2000, J.Biotechnol., 74, 5; Sun, 2000, Cur, Opin. Mol. Ther., 2, 100; Fusser,2000, J. Biotechnol., 74, 27; Hermann and Patel, 2000, Science, 287,820; and Jayasena, 1999, Clinical Chemistry, 45, 1628. Aptamer moleculesof the invention can be chemically modified as is generally known in theart or as described herein.

The term “antisense nucleic acid”, as used herein, refers to a nucleicacid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566)interactions and alters the activity of the target RNA (for a review,see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat.No. 5,849,902) by steric interaction or by RNase H mediated targetrecognition. Typically, antisense molecules are complementary to atarget sequence along a single contiguous sequence of the antisensemolecule. However, in certain embodiments, an antisense molecule canbind to substrate such that the substrate molecule forms a loop, and/oran antisense molecule can bind such that the antisense molecule forms aloop. Thus, the antisense molecule can be complementary to two (or evenmore) non-contiguous substrate sequences or two (or even more)non-contiguous sequence portions of an antisense molecule can becomplementary to a target sequence or both. For a review of currentantisense strategies, see Schmajuk et al., 1999, J. Biol. Chem., 274,21783-21789, Delihas et al., 1997, Nature, 15, 751-753, Stein et al.,1997, Antisense N. A. Drug Dev., 7, 151, Crooke, 2000, Methods Enzymol.,313, 3-45; Crooke, 1998, Biotech genet. Eng. Rev., 15, 121-157, Crooke,1997, Ad. Pharmacol., 40, 1-49. In addition, antisense DNA or antisensemodified with 2′-MOE and other modifications as are known in the art canbe used to target RNA by means of DNA-RNA interactions, therebyactivating RNase H, which digests the target RNA in the duplex. Theantisense oligonucleotides can comprise one or more RNAse H activatingregion, which is capable of activating RNAse H cleavage of a target RNA.Antisense DNA can be synthesized chemically or expressed via the use ofa single stranded DNA expression vector or equivalent thereof Antisensemolecules of the invention can be chemically modified as is generallyknown in the art or as described herein.

By “modulate” is meant that the expression of the gene, or level of aRNA molecule or equivalent RNA molecules encoding one or more proteinsor protein subunits, or activity of one or more proteins or proteinsubunits is up regulated or down regulated, such that expression, level,or activity is greater than or less than that observed in the absence ofthe modulator. For example, the term “modulate” can mean “inhibit,” butthe use of the word “modulate” is not limited to this definition.

By “inhibit”, “down-regulate”, or “reduce”, it is meant that theexpression of the gene, or level of RNA molecules or equivalent RNAmolecules encoding one or more proteins or protein subunits, or activityof one or more proteins or protein subunits, is reduced below thatobserved in the absence of the nucleic acid molecules (e.g., siNA) ofthe invention. In one embodiment, inhibition, down-regulation orreduction with an siNA molecule is below that level observed in thepresence of an inactive or attenuated molecule. In another embodiment,inhibition, down-regulation, or reduction with siNA molecules is belowthat level observed in the presence of, for example, an siNA moleculewith scrambled sequence or with mismatches. In another embodiment,inhibition, down-regulation, or reduction of gene expression with anucleic acid molecule of the instant invention is greater in thepresence of the nucleic acid molecule than in its absence. In oneembodiment, inhibition, down regulation, or reduction of gene expressionis associated with post transcriptional silencing, such as RNAi mediatedcleavage of a target nucleic acid molecule (e.g., RNA) or inhibition oftranslation. In one embodiment, inhibition, down regulation, orreduction of gene expression is associated with pretranscriptionalsilencing, such as by alterations in DNA methylation patterns and DNAchromatin structure.

By “up-regulate”, or “promote”, it is meant that the expression of thegene, or level of RNA molecules or equivalent RNA molecules encoding oneor more proteins or protein subunits, or activity of one or moreproteins or protein subunits, is increased above that observed in theabsence of the nucleic acid molecules siNA) of the invention. In oneembodiment, up-regulation or promotion of gene expression with an siNAmolecule is above that level observed in the presence of an inactive orattenuated molecule. In another embodiment, up-regulation or promotionof gene expression with siNA molecules is above that level observed inthe presence of, for example, an siNA molecule with scrambled sequenceor with mismatches. In another embodiment, up-regulation or promotion ofgene expression with a nucleic acid molecule of the instant invention isgreater in the presence of the nucleic acid molecule than in itsabsence. In one embodiment, up-regulation or promotion of geneexpression is associated with inhibition of RNA mediated gene silencing,such as RNAi mediated cleavage or silencing of a coding or non-codingRNA target that down regulates, inhibits, or silences the expression ofthe gene of interest to be up-regulated. The down regulation of geneexpression can, for example, be induced by a coding RNA or its encodedprotein, such as through negative feedback or antagonistic effects. Thedown regulation of gene ‘expression can, for example, be induced by anon-coding RNA having regulatory control over a gene of interest, forexample by silencing expression of the gene via translationalinhibition, chromatin structure, methylation, RISC mediated RNAcleavage, or translational inhibition. As such, inhibition or downregulation of targets that down regulate, suppress, or silence a gene ofinterest can be used to up-regulate or promote expression of the gene ofinterest toward therapeutic use.

In one embodiment, a RNAi inhibitor of the invention is used to upregulate gene expression by inhibiting RNAi or gene silencing. Forexample, a RNAi inhibitor of the invention can be used to treat loss offunction diseases and conditions by up-regulating gene expression, suchas in instances of haploinsufficiency where one allele of a particulargene harbors a mutation (e.g., a frameshift, missense, or nonsensemutation) resulting in a loss of function of the protein encoded by themutant allele. In such instances, the RNAi inhibitor can be used to upregulate expression of the protein encoded by the wild type orfunctional allele, thus correcting the haploinsufficiency bycompensating for the mutant or null allele. In another embodiment, asiNA molecule of the invention is used to down regulate expression of atoxic gain of function allele while a RNAi inhibitor of the invention isused concomitantly to up regulate expression of the wild type orfunctional allele, such as in the treatment of diseases, traits, orconditions herein or otherwise known in the art (see for example Rhodeset al., 2004, PNAS USA, 101:11147-11152 and Meisler et al. 2005, TheJournal of Clinical Investigation, 115:2010-2017).

By “gene”, or “target gene” or “target DNA”, is meant a nucleic acidthat encodes an RNA, for example, nucleic acid sequences including, butnot limited to, structural genes encoding a polypeptide. A gene ortarget gene can also encode a functional RNA (fRNA) or non-coding RNA(ncRNA), such as small temporal RNA (stRNA), micro RNA (miRNA), smallnuclear RNA (snRNA), short interfering RNA (siRNA), small nucleolar RNA(snRNA), ribosomal RNA (rRNA), transfer RNA (tRNA) and precursor RNAsthereof Such non-coding RNAs can serve as target nucleic acid moleculesfor siNA mediated RNA interference in modulating the activity of fRNA orncRNA involved in functional or regulatory cellular processes. AbberantfRNA or ncRNA activity leading to disease can therefore be modulated bysiNA molecules of the invention. siNA molecules targeting fRNA and ncRNAcan also be used to manipulate or alter the genotype or phenotype of asubject, organism or cell, by intervening in cellular processes such asgenetic imprinting, transcription, translation, or nucleic acidprocessing (e.g., transamination, methylation etc.). The target gene canbe a gene derived from a cell, an endogenous gene, a transgene, orexogenous genes such as genes of a pathogen, for example a virus, whichis present in the cell after infection thereof. The cell containing thetarget gene can be derived from or contained in any organism, forexample a plant, animal, protozoan, virus, bacterium, or fungus.Non-limiting examples of plants include monocots, dicots, orgymnosperms. Non-limiting examples of animals include vertebrates orinvertebrates. Non-limiting examples of fungi include molds or yeasts.For a review, see for example Snyder and Gerstein, 2003, Science, 300,258-260.

By “non-canonical base pair” is meant any non-Watson Crick base pair,such as mismatches and/or wobble base pairs, including flippedmismatches, single hydrogen bond mismatches, trans-type mismatches,triple base interactions, and quadruple base interactions. Non-limitingexamples of such non-canonical base pairs include, but are not limitedto, AC reverse Hoogsteen, AC wobble, AU reverse Hoogsteen, GU wobble, AAN7 amino, CC 2-carbonyl-amino(H1)-N3-amino(H2), GA sheared, UC4-carbonyl-amino, UU imino-carbonyl, AC reverse wobble, AU Hoogsteen, AUreverse Watson Crick, CG reverse Watson Crick, GC N3-amino-amino N3, AAN1-amino symmetric, AA N7-amino symmetric, GA N7-N1 amino-carbonyl, GA+carbonyl-amino N7-N1, GG N1-carbonyl symmetric, GG N3-amino symmetric,CC carbonyl-amino symmetric, CC N3-amino symmetric, UU 2-carbonyl-iminosymmetric, UU 4-carbonyl-imino symmetric, AA amino-N3, AA N1-amino, ACamino 2-carbonyl, AC N3-amino, AC N7-amino, AU amino-4-carbonyl, AUN1-imino, AU N3-imino, AU N7-imino, CC carbonyl-amino, GA amino-N1, GAamino-N7, GA carbonyl-amino, GA N3-amino, GC amino-N3, GCcarbonyl-amino, GC N3-amino, GC N7-amino, GG amino-N7, GGcarbonyl-imino, GG N7-amino, GU amino-2-carbonyl, GU carbonyl-imino, GUimino-2-carbonyl, GU N7-imino, psiU imino-2-carbonyl, UC4-carbonyl-amino, UC imino-carbonyl, UU imino-4-carbonyl, AC C2-H-N3, GAcarbonyl-C2-H, UU imino-4-carbonyl 2 carbonyl-05-H, AC amino(A)N3(C)-carbonyl, GC imino amino-carbonyl, Gpsi imino-2-carbonylamino-2-carbonyl, and GU imino amino-2-carbonyl base pairs.

By “target” as used herein is meant, any target protein, peptide, orpolypeptide, such as encoded by Genbank Accession Nos. described hereinand/or in U.S. Provisional Patent Application No. 60/363,124, U.S. Ser.No. 10/923,536 and/or PCl/US03/05028, both incorporated by referenceherein. The term “target” also refers to nucleic acid sequences ortarget polynucleotide sequence encoding any target protein, peptide, orpolypeptide, such as proteins, peptides, or polypeptides encoded bysequences having Genbank Accession Nos. shown herein and/or in U.S.Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536and/or USSN PCT/US03/05028. The target of interest can include targetpolynucleotide sequences, such as target DNA or target RNA. The term“target” is also meant to include other sequences, such as differingisoforms, mutant target genes, splice variants of targetpolynucleotides, target polymorphisms, and non-coding (e.g., ncRNA,miRNA, stRNA) or other regulatory polynucleotide sequences as describedherein. Therefore, in various embodiments of the invention, a doublestranded nucleic acid molecule of the invention (e.g., siNA) havingcomplementarity to a target RNA can be used to inhibit or down regulatemiRNA or other ncRNA activity. In one embodiment, inhibition of miRNA orncRNA activity can be used to down regulate or inhibit gene expression(e.g., gene targets described herein or otherwise known in the art) thatis dependent on miRNA or ncRNA activity. In another embodiment,inhibition of miRNA or ncRNA activity by double stranded nucleic acidmolecules of the invention (e.g., siNA) having complementarity to themiRNA or ncRNA can be used to up regulate or promote target geneexpression (e.g., gene targets described herein or otherwise known inthe art) where the expression of such genes is down regulated,suppressed, or silenced by the miRNA or ncRNA. Such up-regulation ofgene expression can be used to treat diseases and conditions associatedwith a loss of function or haploinsufficiency as are generally known inthe art (e.g., muscular dystrophies, cystic fibrosis, or neurologicdiseases and conditions described herein such as epilepsy, includingsevere myoclonic epilepsy of infancy or Dravet syndrome).

By “pathway target” is meant any target involved in pathways of geneexpression or activity. For example, any given target can have relatedpathway targets that can include upstream, downstream, or modifier genesin a biologic pathway. These pathway target genes can provide additiveor synergistic effects in the treatment of diseases, conditions, andtraits herein.

In one embodiment, the target is any of target RNA or a portion thereof.

In one embodiment, the target is any target DNA or a portion thereof.

In one embodiment, the target is any target mRNA or a portion thereof.

In one embodiment, the target is any target miRNA or a portion thereof.

In one embodiment, the target is any target siRNA or a portion thereof.

In one embodiment, the target is any target stRNA or a portion thereof.

In one embodiment, the target is a target and or pathway target or aportion thereof.

In one embodiment, the target is any (e.g., one or more) of targetsequences described herein and/or in U.S. Provisional Patent ApplicationNo. 60/363,124, U.S. Ser. No. 10/923,536 and/or PCT/US03/05028, or aportion thereof. In one embodiment, the target is any (e.g., one ormore) of target sequences shown in Table II or a portion thereof. Inanother embodiment, the target is a siRNA, miRNA, or stRNA correspondingto any (e.g., one or more) target, upper strand, or lower strandsequence shown in Table II or a portion thereof. In another embodiment,the target is any siRNA, miRNA, or stRNA corresponding any (e.g., one ormore) sequence corresponding to a sequence herein or described in U.S.Provisional Patent Application No. 60/363,124, U.S. Ser. No. 10/923,536and/or PC T/US 03/05028.

By “homologous sequence” is meant, a nucleotide sequence that is sharedby one or more polynucleotide sequences, such as genes, gene transcriptsand/or non-coding polynucleotides. For example, a homologous sequencecan be a nucleotide sequence that is shared by two or more genesencoding related but different proteins, such as different members of agene family, different protein epitopes, different protein isoforms orcompletely divergent genes, such as a cytokine and its correspondingreceptors. A homologous sequence can be a nucleotide sequence that isshared by two or more non-coding polynucleotides, such as noncoding DNAor RNA, regulatory sequences, introns, and sites of transcriptionalcontrol or regulation. Homologous sequences can also include conservedsequence regions shared by more than one polynucleotide sequence.Homology does not need to be perfect homology (e.g., 100%), as partiallyhomologous sequences are also contemplated by the instant invention(e.g., 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%,86%, 85%, 84%, 83%, 82%, 81%, 80% etc.).

By “conserved sequence region” is meant, a nucleotide sequence of one ormore regions in a polynucleotide does not vary significantly betweengenerations or from one biological system, subject, or organism toanother biological system, subject, or organism. The polynucleotide caninclude both coding and non-coding DNA and RNA.

By “sense region” is meant a nucleotide sequence of a siNA moleculehaving complementarity to an antisense region of the siNA molecule. Inaddition, the sense region of a siNA molecule can comprise a nucleicacid sequence having homology with a target nucleic acid sequence. Inone embodiment, the sense region of the siNA molecule is referred to asthe sense strand or passenger strand.

By “antisense region” is meant a nucleotide sequence of a siNA moleculehaving complementarily to a target nucleic acid sequence. In addition,the antisense region of a siNA molecule can optionally comprise anucleic acid sequence having complementarity to a sense region of thesiNA molecule. In one embodiment, the antisense region of the siNAmolecule is referred to as the antisense strand or guide strand.

By “target nucleic acid” or “target polynucleotide” is meant any nucleicacid sequence (e.g, any target and/or pathway target sequence) whoseexpression or activity is to be modulated. The target nucleic acid canbe DNA or RNA. In one embodiment, a target nucleic acid of the inventionis target RNA or DNA.

By “complementarity” is meant that a nucleic acid can form hydrogenbond(s) with another nucleic acid sequence by either traditionalWatson-Crick or other non-traditional types as described herein. In oneembodiment, a double stranded nucleic acid molecule of the invention,such as an siNA molecule, wherein each strand is between 15 and 30nucleotides in length, comprises between about 10% and about 100% (e.g.,about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%)complementarity between the two strands of the double stranded nucleicacid molecule. In another embodiment, a double stranded nucleic acidmolecule of the invention, such as an siNA molecule, where one strand isthe sense strand and the other stand is the antisense strand, whereineach strand is between 15 and 30 nucleotides in length, comprisesbetween at least about 10% and about 100% (e.g., at least about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity betweenthe nucleotide sequence in the antisense strand of the double strandednucleic acid molecule and the nucleotide sequence of its correspondingtarget nucleic acid molecule, such as a target RNA or target mRNA orviral RNA. In one embodiment, a double stranded nucleic acid molecule ofthe invention, such as an siNA molecule, where one strand comprisesnucleotide sequence that is referred to as the sense region and theother strand comprises a nucleotide sequence that is referred to as theantisense region, wherein each strand is between 15 and 30 nucleotidesin length, comprises between about 10% and about 100% (e.g., about 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) complementarity betweenthe sense region and the antisense region of the double stranded nucleicacid molecule. In reference to the nucleic molecules of the presentinvention, the binding free energy for a nucleic acid molecule with itscomplementary sequence is sufficient to allow the relevant function ofthe nucleic acid to proceed, e.g., RNAi activity. Determination ofbinding free energies for nucleic acid molecules is well known in theart (see, e.g., Turner et al., 1987, CSH Symp. Quant. Biol. LII pp.123-133; Frier et al., 1986, Proc. Nat. Acad. Sci. USA 83:9373-9377;Turner et al., 1987, J. Am. Chem. Soc. 109:3783-3785). A percentcomplementarity indicates the percentage of contiguous residues in anucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,or 10 nucleotides out of a total of 10 nucleotides in the firstoligonucleotide being based paired to a second nucleic acid sequencehaving 10 nucleotides represents 50%, 60%, 70%, 80%, 90%, and 100%complementary respectively). In one embodiment, a siNA molecule of theinvention has perfect complementarity between the sense strand or senseregion and the antisense strand or antisense region of the siNAmolecule. In one embodiment, a siNA molecule of the invention isperfectly complementary to a corresponding target nucleic acid molecule.“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. In oneembodiment, a siNA molecule of the invention comprises about 15 to about30 or more (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, or 30 or more) nucleotides that are complementary to one ormore target nucleic acid molecules or a portion thereof. In oneembodiment, a siNA molecule of the invention has partial complementarity(i.e., less than 100% complementarity) between the sense strand or senseregion and the antisense strand or antisense region of the siNA moleculeor between the antisense strand or antisense region of the siNA moleculeand a corresponding target nucleic acid molecule. For example, partialcomplementarity can include various mismatches or non-based pairednucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based pairednucleotides) within the siNA structure which can result in bulges,loops, or overhangs that result between the between the sense strand orsense region and the antisense strand or antisense region of the siNAmolecule or between the antisense strand or antisense region of the siNAmolecule and a corresponding target nucleic acid molecule.

In one embodiment, a double stranded nucleic acid molecule of theinvention, such as siNA molecule, has perfect complementarity betweenthe sense strand or sense region and the antisense strand or antisenseregion of the nucleic acid molecule. In one embodiment, double strandednucleic acid molecule of the invention, such as siNA molecule, isperfectly complementary to a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of theinvention, such as siNA molecule, has partial complementarity (i.e.,less than 100% complementarity) between the sense strand or sense regionand the antisense strand or antisense region of the double strandednucleic acid molecule or between the antisense strand or antisenseregion of the nucleic acid molecule and a corresponding target nucleicacid molecule. For example, partial complementarity can include variousmismatches or non-base paired nucleotides (e.g., 1, 2, 3, 4, 5 or moremismatches or non-based paired nucleotides, such as nucleotide bulges)within the double stranded nucleic acid molecule, structure which canresult in bulges, loops, or overhangs that result between the sensestrand or sense region and the antisense strand or antisense region ofthe double stranded nucleic acid molecule or between the antisensestrand or antisense region of the double stranded nucleic acid moleculeand a corresponding target nucleic acid molecule.

In one embodiment, double stranded nucleic acid molecule of theinvention is a microRNA (miRNA). By “microRNA” or “miRNA” is meant, asmall double stranded RNA that regulates the expression of targetmessenger RNAs either by mRNA cleavage; translationalrepression/inhibition or heterochromatic silencing (see for exampleAmbros, 2004, Nature, 431, 350-355; Bartel, 2004, Cell, 116, 281-297;Cullen, 2004, Virus Research., 102, 3-9; He et al., 2004, Nat. Rev.genet., 5, 522-531; Ying et al., 2004, gene, 342, 25-28; and Sethupathyet al., 2006, RNA, 12:192-197). In one embodiment, the microRNA of theinvention, has partial complementarity (i.e., less than 100%complementarity) between the sense strand or sense region and theantisense strand or antisense region of the miRNA molecule or betweenthe antisense strand or antisense region of the miRNA and acorresponding target nucleic acid molecule. For example, partialcomplementarity can include various mismatches or non-base pairednucleotides (e.g., 1, 2, 3, 4, 5 or more mismatches or non-based pairednucleotides, such as nucleotide bulges) within the double strandednucleic acid molecule, structure which can result in bulges, loops, oroverhangs that result between the sense strand or sense region and theantisense strand or antisense region of the miRNA or between theantisense strand or antisense region of the miRNA and a correspondingtarget nucleic acid molecule.

In one embodiment, siNA molecules of the invention that down regulate orreduce target gene expression are used for preventing or treatingdiseases, disorders, conditions, or traits in a subject or organism asdescribed herein or otherwise known in the art.

By “proliferative disease” or “cancer” as used herein is meant, anydisease, condition, trait, genotype or phenotype characterized byunregulated cell growth or replication as is known in the art; includingleukemias, for example, acute myelogenous leukemia (AML), chronicmyelogenous leukemia (CML), acute lymphocytic leukemia (ALL), andchronic lymphocytic leukemia, AIDS related cancers such as Kaposi'ssarcoma; breast cancers; bone cancers such as Osteosarcoma,Chondrosarcomas, Ewing's sarcoma, Fibrosarcomas, Giant cell tumors,Adamantinomas, and Chordomas; Brain cancers such as Meningiomas,Glioblastomas, Lower-Grade Astrocytomas, Oligodendrocytomas, PituitaryTumors, Schwannomas, and Metastatic brain cancers; cancers of the headand neck including various lymphomas such as mantle cell lymphoma,non-Hodgkins lymphoma, adenoma, squamous cell carcinoma, laryngealcarcinoma, gallbladder and bile duct cancers, cancers of the retina suchas retinoblastoma, cancers of the esophagus, gastric cancers, multiplemyeloma, ovarian cancer, uterine cancer, thyroid cancer, testicularcancer, endometrial cancer, melanoma, colorectal cancer, lung cancer,bladder cancer, prostate cancer, lung cancer (including non-small celllung carcinoma), pancreatic cancer, sarcomas, Wilms' tumor, cervicalcancer, head and neck cancer, skin cancers, nasopharyngeal carcinoma,liposarcoma, epithelial carcinoma, renal cell carcinoma, gallbladderadeno carcinoma, parotid adenocarcinoma, endometrial sarcoma, multidrugresistant cancers; and proliferative diseases and conditions, such asneovascularization associated with tumor angiogenesis, maculardegeneration (e.g., wet/dry AMD), corneal neovascularization, diabeticretinopathy, neovascular glaucoma, myopic degeneration and otherproliferative diseases and conditions such as restenosis and polycystickidney disease, and any other cancer or proliferative disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “inflammatory disease” or “inflammatory condition” as used herein ismeant any disease, condition, trait, genotype or phenotype characterizedby an inflammatory or allergic process as is known in the art, such asinflammation, acute inflammation, chronic inflammation, respiratorydisease, atherosclerosis, psoriasis, dermatitis, restenosis, asthma,allergic rhinitis, atopic dermatitis, septic shock, rheumatoidarthritis, inflammatory bowl disease, inflammatory pelvic disease, pain,ocular inflammatory disease, celiac disease, Leigh Syndrome, GlycerolKinase Deficiency, Familial eosinophilia (FE), autosomal recessivespastic ataxia, laryngeal inflammatory disease; Tuberculosis, Chroniccholecystitis, Bronchiectasis, Silicosis and other pneumoconioses, andany other inflammatory disease, condition, trait, genotype or phenotypethat can respond to the modulation of disease related gene expression ina cell or tissue, alone or in combination with other therapies.

By “autoimmune disease” or “autoimmune condition” as used herein ismeant, any disease, condition, trait, genotype or phenotypecharacterized by autoimmunity as is known in the art, such as multiplesclerosis, diabetes mellitus, lupus, celiac disease, Crohn's disease,ulcerative colitis, Guillain-Barre syndrome, scleroderms, Goodpasture'ssyndrome, Wegener's granulomatosis, autoimmune epilepsy, Rasmussen'sencephalitis, Primary biliary sclerosis, Sclerosing cholangitis,Autoimmune hepatitis, Addison's disease, Hashimoto's thyroiditis,Fibromyalgia, Menier's syndrome; transplantation rejection (e.g.,prevention of allograft rejection) pernicious anemia, rheumatoidarthritis, systemic lupus erythematosus, dermatomyositis, Sjogren'ssyndrome, lupus erythematosus, multiple sclerosis, myasthenia gravis,Reiter's syndrome, Grave's disease, and any other autoimmune disease,condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “infectious disease” is meant any disease, condition, trait, genotypeor phenotype associated with an infectious agent, such as a virus,bacteria, fungus, prion, or parasite. Non-limiting examples of variousviral genes that can be targeted using siNA molecules of the inventioninclude Hepatitis C Virus (HCV, for example Genbank Accession Nos:D11168, D50483.1, L38318 and S82227), Hepatitis B Virus (HBV, forexample GenBank Accession No. AF100308.1), Human Immunodeficiency Virustype 1 (HIV-1, for example GenBank Accession No. U51188), HumanImmunodeficiency Virus type 2 (HIV-2, for example GenBank Accession No.X60667), West Nile Virus (WNV for example GenBank accession No.NC_001563), cytomegalovirus (CMV for example GenBank Accession No.NC_001347), respiratory syncytial virus (RSV for example GenBankAccession No. NC_001781), influenza virus (for example GenBank AccessionNo. AF037412, rhinovirus (for example, GenBank accession numbers:D00239, X02316, X01087, L24917, M16248, K02121, X01087), papillomavirus(for example GenBank Accession No. NC_001353), Herpes Simplex Virus (HSVfor example GenBank Accession No. NC_001345), and other viruses such asHTLV (for example GenBank Accession No. AJ430458). Due to the highsequence variability of many viral genomes, selection of siNA moleculesfor broad therapeutic applications would likely involve the conservedregions of the viral genome. Nonlimiting examples of conserved regionsof the viral genomes include but are not limited to 5′-Non CodingRegions (NCR), 3′-Non Coding Regions (NCR) and/or internal ribosomeentry sites (IRES). siNA molecules designed against conserved regions ofvarious viral genomes will enable efficient inhibition of viralreplication in diverse patient populations and may ensure theeffectiveness of the siNA molecules against viral quasi species whichevolve due to mutations in the non-conserved regions of the viralgenome. Non-limiting examples of bacterial infections includeActinomycosis, Anthrax, Aspergillosis, Bacteremia, Bacterial Infectionsand Mycoses, Bartonella Infections, Botulism, Brucellosis, BurkholderiaInfections, Campylobacter Infections, Candidiasis, Cat-Scratch Disease,Chlamydia Infections, Cholera, Clostridium Infections,Coccidioidomycosis, Cross Infection, Cryptococcosis, Dermatomycoses,Dermatomycoses, Diphtheria, Ehrlichiosis, Escherichia coli Infections,Fasciitis, Necrotizing, Fusobacterium Infections, Gas Gangrene,Gram-Negative Bacterial Infections, Gram-Positive Bacterial Infections,Histoplasmosis, Impetigo, Klebsiella Infections, Legionellosis, Leprosy,Leptospirosis, Listeria Infections, Lyme Disease, Maduromycosis,Melioidosis, Mycobacterium Infections, Mycoplasma Infections, Mycoses,Nocardia Infections, Onychomycosis, Ornithosis, Plague, PneumococcalInfections, Pseudomonas Infections, Q Fever, Rat-Bite Fever, RelapsingFever, Rheumatic Fever, Rickettsia Infections, Rocky Mountain SpottedFever, Salmonella Infections, Scarlet Fever, Scrub Typhus, Sepsis,Sexually Transmitted Diseases—Bacterial, Bacterial Skin Diseases,Staphylococcal Infections, Streptococcal Infections, Tetanus, Tick-BorneDiseases, Tuberculosis, Tularemia, Typhoid Fever, Typhus, EpidemicLouse-Borne, Vibrio Infections, Yaws, Yersinia Infections, Zoonoses, andZygomycosis. Non-limiting examples of fungal infections includeAspergillosis, Blastomycosis, Coccidioidomycosis, Cryptococcosis, FungalInfections of Fingernails and Toenails, Fungal Sinusitis,Histoplasmosis, Histoplasmosis, Muconnycosis, Nail Fungal Infection,Paracoccidioidomycosis, Sporotrichosis, Valley Fever(Coccidioidomycosis), and Mold Allergy.

By “neurologic disease” or “neurological disease” is meant any disease,disorder, or condition affecting the central or peripheral nervoussystem, inlcuding ADFID, AIDS—Neurological Complications, Absence of theSeptum Pellucidum, Acquired Epileptiform Aphasia, Acute DisseminatedEncephalomyelitis, Adrenoleukodystrophy, Agenesis of the CorpusCallosum, Agnosia, Aicardi Syndrome, Alexander Disease, Alpers' Disease,Alternating Hemiplegia, Alzheimer's Disease, Amyotrophic LateralSclerosis, Anencephaly, Aneurysm, Angelman Syndrome, Angiomatosis,Anoxia, Aphasia, Apraxia, Arachnoid Cysts, Arachnoiditis, Arnold-ChiariMalformation, Arteriovenous Malformation, Aspartame, Asperger Syndrome,Ataxia Telangiectasia, Ataxia, Attention Deficit-Hyperactivity Disorder,Autism, Autonomic Dysfunction, Back Pain, Barth Syndrome, BattenDisease, Behcet's Disease, Bell's Palsy, Benign Essential Blepharospasm,Benign Focal Amyotrophy, Benign Intracranial Hypertension,Bernhardt-Roth Syndrome, Binswanger's Disease, Blepharospasm,Bloch-Sulzberger Syndrome, Brachial Plexus Birth Injuries, BrachialPlexus Injuries, Bradbury-Eggleston Syndrome, Brain Aneurysm, BrainInjury, Brain and Spinal Tumors, Brown-Sequard Syndrome, BulbospinalMuscular Atrophy, Canavan Disease, Carpal Tunnel Syndrome, Causalgia,Cavernomas, Cavernous Angioma, Cavernous Malformation, Central CervicalCord Syndrome, Central Cord Syndrome, Central Pain Syndrome, CephalicDisorders, Cerebellar Degeneration, Cerebellar Hypoplasia, CerebralAneurysm, Cerebral Arterioscicrosis, Cerebral Atrophy, CerebralBeriberi, Cerebral Gigantism, Cerebral Hypoxia, Cerebral Palsy,Cerebro-Oculo-Facio-Skeletal Syndrome, Charcot-Marie-Tooth Disorder,Chiari Malformation, Chorea, Choreoacanthocytosis, Chronic InflammatoryDemyelinating Polyneuropathy (CIDP), Chronic Orthostatic Intolerance,Chronic Pain, Cockayne Syndrome Type II, Coffin Lowry Syndrome, Coma,including Persistent Vegetative State, Complex Regional Pain Syndrome,Congenital Facial Diplegia, Congenital Myasthenia, Congenital Myopathy,Congenital Vascular Cavernous Malformations, Corticobasal Degeneration,Cranial Arteritis, Craniosynostosis, Creutzfeldt-Jakob Disease,Cumulative Trauma Disorders, Cushing's Syndrome, Cytomegalic InclusionBody Disease (CIBD), Cytomegalovirus Infection, Dancing Eycs-DancingFeet Syndrome, Dandy-Walker Syndrome, Dawson Disease, De Morsier'sSyndrome, Dejerine-Klumpke Palsy, Dementia—Multi-Infarct,Dementia—Subcortical, Dementia With Lewy Bodies, Dermatomyositis,Developmental Dyspraxia, Devic's Syndrome, Diabetic Neuropathy, DiffuseSclerosis, Dravet's Syndrome, Dysautonomia, Dysgraphia, Dyslexia,Dysphagia, Dyspraxia, Dystonias, Early Infantile EpilepticEncephalopathy, Empty Sella Syndrome, Encephalitis Lethargica,Encephalitis and Meningitis, Encephaloceles, Encephalopathy,Encephalotrigeminal Angiomatosis, Epilepsy, Erb's Palsy, Erb-Duehenneand Dejerine-Klumpke Palsies, Fabry's Disease, Fahr's Syndrome,Fainting, Familial Dysautonomia, Familial Hemangioma, FamilialIdiopathic Basal Ganglia Calcification, Familial Spastic Paralysis,Febrile Seizures (e.g., GEFS and GEFS plus), Fisher Syndrome, FloppyInfant Syndrome, Friedreich's Ataxia, Gaucher's Disease, Gerstmann'sSyndrome, Gerstmann-Straussler-Scheinker Disease, Giant Cell Arteritis,Giant Cell Inclusion Disease, Globoid Cell Leukodystrophy,Glossopharyngeal Neuralgia, Guillain-Barre Syndrome, HTLV-1 AssociatedMyelopathy, Hallervorden-Spatz Disease, Head Injury, Headache,Hemicrania Continua, Hemifacial Spasm, Hemiplegia Alterans, HereditaryNeuropathies, Hereditary Spastic Paraplegia, Heredopathia AtacticaPolyneuritiformis, Herpes Zoster Oticus, Herpes Zoster, HirayamaSyndrome, Holoprosencephaly, Huntington's Disease, Hydranencephaly,Hydrocephalus—Normal Pressure, Hydrocephalus, Hydromyelia,Hypercortisolism, Hypersomnia, Hypertonia, Hypotonia, Hypoxia,Immune-Mediated Encephalomyelitis, Inclusion Body Myositis,Incontinentia Pigmenti, Infantile Hypotonia, Infantile Phytanic AcidStorage Disease, Infantile Refsum Disease, Infantile Spasms,Inflammatory Myopathy, Intestinal Lipodystrophy, Intracranial Cysts,Intracranial Hypertension, Isaac's Syndrome, Joubert Syndrome,Kearns-Sayre Syndrome, Kennedy's Disease, Kinsbourne syndrome,Kleine-Levin syndrome, Klippel Feil Syndrome, Klippel-Trenaunay Syndrome(KTS), Kluver-Bucy Syndrome, Korsakoffs Amnesic Syndrome, KrabbeDisease, Kugelberg-Welander Disease, Kuru, Lambert-Eaton MyasthenieSyndrome, Landau-Kleffner Syndrome, Lateral Femoral Cutaneous NerveEntrapment, Lateral Medullary Syndrome, Learning Disabilities, Leigh'sDisease, Lennox-Gastaut Syndrome, Lesch-Nyhan Syndrome, Leukodystrophy,Levine-Critchley Syndrome, Lewy Body Dementia, Lissencephaly, Locked-InSyndrome, Lou Gehrig's Disease, Lupus—Neurological Sequelae, LymeDisease—Neurological Complications, Machado-Joseph Disease,Macrencephaly, Megalencephaly, Melkersson-Rosenthal Syndrome,Meningitis, Menkes Disease, Meralgia Paresthetica, MetachromaticLeukodystrophy, Microcephaly, Migraine, Miller Fisher Syndrome,Mini-Strokes, Mitochondrial Myopathies, Mobius Syndrome, MonomelicAmyotrophy, Motor Neuron Diseases, Moyamoya Disease, Mucolipidoses,Mucopolysaccharidoses; Multi-Infarct Dementia, Multifocal MotorNeuropathy, Multiple Sclerosis, Multiple System Atrophy with OrthostaticHypotension, Multiple System Atrophy, Muscular Dystrophy,Myasthenia—Congenital, Myasthenia Gravis, Myelinoclastic DiffuseSclerosis, Myoclonic Encephalopathy of Infants, Myoclonus,Myopathy—Congenital, Myopathy Thyrotoxic, Myopathy, Myotonia Congenita,Myotonia, Narcolepsy, Neuroacanthocytosis, Neurodegeneration with BrainIron Accumulation, Neurofibromatosis, Neuroleptic Malignant Syndrome,Neurological Complications of AIDS, Neurological Manifestations of PompeDisease, Neuromyelitis Optica, Neuromyotonia, Neuronal CeroidLipofuscinosis, Neuronal Migration Disorders, Neuropathy—Hereditary,Neurosarcoidosis, Neurotoxicity, Nevus Cavernosus, Niemann-Pick Disease,O'Sullivan-McLeod Syndrome, Occipital Neuralgia, Occult SpinalDysraphism Sequence, Ohtahara Syndrome, Olivopontocerebellar Atrophy,Opsoclonus Myoclonus, Orthostatic Hypotension, Overuse Syndrome,Pain—Chronic, Paraneoplastic Syndromes, Paresthesia, Parkinson'sDisease, Parmyotonia Congenita, Paroxysmal Choreoathetosis, ParoxysmalHemicrania, Parry-Romberg, Pelizaeus-Merzbacher Disease, Pena Shokeir IISyndrome, Perineural Cysts, Periodic Paralyses, Peripheral Neuropathy,Periventricular Leukomalacia, Persistent Vegetative State, PervasiveDevelopmental Disorders, Phytanic Acid Storage Disease, Pick's Disease,Piriformis Syndrome, Pituitary Tumors, Polymyositis, Pompe Disease,Porencephaly, Post-Polio Syndrome, Postherpetic Neuralgia,Postinfectious Encephalomyelitis, Postural Hypotension, PosturalOrthostatic Tachycardia Syndrome, Postural Tachycardia Syndrome, PrimaryLateral Sclerosis, Prion Diseases, Progressive Hemifacial Atrophy,Progressive Locomotor Ataxia, Progressive MultifocalLeukoencephalopathy, Progressive Sclerosing Poliodystrophy, ProgressiveSupranuclear Palsy, Pseudotumor Cerebri, Pyridoxine Dependent andPyridoxine Responsive Siezure Disorders, Ramsay Hunt Syndrome Type I,Ramsay Hunt Syndrome Type II, Rasmussen's Encephalitis and otherautoimmune epilepsies, Reflex Sympathetic DyStrophy Syndrome, RefsumDisease—Infantile, Refsum Disease, Repetitive Motion Disorders,Repetitive Stress Injuries, Restless Legs Syndrome,Retrovirus-Associated Myelopathy, Rett Syndrome, Reye's Syndrome,Riley-Day Syndrome, SUNCT Headache, Sacral Nerve Root Cysts, Saint VitusDance, Salivary Gland Disease, Sandhoff Disease, Schilder's Disease,Schizencephaly, Seizure Disorders, Septo-Optic Dysplasia, SevereMyoclonic Epilepsy of Infancy (SMEI), Shaken Baby Syndrome, Shingles,Shy-Drager Syndrome, Sjogren's Syndrome, Sleep Apnea, Sleeping Sickness,Soto's Syndrome, Spasticity, Spina Bifida, Spinal Cord Infarction,Spinal Cord Injury, Spinal Cord Tumors, Spinal Muscular Atrophy,Spinocerebellar Atrophy, Steele-Richardson-Olszewski Syndrome,Stiff-Person Syndrome, Striatonigral Degeneration, Stroke, Sturge-WeberSyndrome, Subacute Sclerosing Panencephalitis, SubcorticalArteriosclerotic Encephalopathy, Swallowing Disorders, Sydenham Chorea,Syncope, Syphilitic Spinal Sclerosis, Syringohydromyelia, Syringomyelia,Systemic Lupus Erythematosus, Tabes Dorsalis, Tardive Dyskinesia, TarlovCysts, Tay-Sachs Disease, Temporal Arteritis, Tethered Spinal CordSyndrome, Thomsen Disease, Thoracic Outlet Syndrome, ThyrotoxicMyopathy, Tic Douloureux, Todd's Paralysis, Tourette Syndrome, TransientIschemic Attack, Transmissible Spongiform Encephalopathies, TransverseMyelitis, Traumatic Brain Injury, Tremor, Trigeminal Neuralgia, TropicalSpastic Paraparesis, Tuberous Sclerosis, Vascular Erectile Tumor,Vasculitis including Temporal Arteritis, Von Economo's Disease, VonHippel-Lindau disease (VHL), Von Recklinghausen's Disease, Wallenberg'sSyndrome, Werdnig-Hoffman Disease, Wernicke-Korsakoff Syndrome, WestSyndrome, Whipple's Disease, Williams Syndrome, Wilson's Disease,X-Linked Spinal and Bulbar Muscular Atrophy, and Zellweger Syndrome.

By “respiratory disease” is meant, any disease or condition affectingthe respiratory tract, such as asthma, chronic obstructive pulmonarydisease or “COPD”, allergic rhinitis, sinusitis, pulmonaryvasoconstriction, inflammation, allergies, impeded respiration,respiratory distress syndrome, cystic fibrosis, pulmonary hypertension,pulmonary vasoconstriction, emphysema, and any other respiratorydisease, condition, trait, genotype or phenotype that can respond to themodulation of disease related gene expression in a cell or tissue, aloneor in combination with other therapies.

By “ocular disease” as used herein is meant, any disease, condition,trait, genotype or phenotype of the eye and related structures as isknown in the art, such as Cystoid Macular Edema, Asteroid Hyalosis,Pathological Myopia and Posterior Staphyloma, Toxocariasis (Ocular LarvaMigrans), Retinal Vein Occlusion, Posterior Vitreous Detachment,Tractional Retinal Tears, Epiretinal Membrane, Diabetic Retinopathy,Lattice Degeneration, Retinal Vein Occlusion, Retinal Artery Occlusion,Macular Degeneration (e.g., age related macular degeneration such as wetAMD or dry AMD), Toxoplasmosis, Choroidal Melanoma, AcquiredRetinoschisis, Hollenhorst Plaque, Idiopathic Central SerousChorioretinopathy, Macular Hole, Presumed Ocular HistoplasmosisSyndrome, Retinal Macroaneursym, Retinitis Pigmentosa, RetinalDetachment, Hypertensive Retinopathy, Retinal Pigment Epithelium (RPE)Detachment, Papillophlebitis, Ocular Ischemic Syndrome, Coats' Disease,Leber's Miliary Aneurysm, Conjunctival Neoplasms, AllergicConjunctivitis, Vernal Conjunctivitis, Acute Bacterial Conjunctivitis,Allergic Conjunctivitis &Vernal Keratoconjunctivitis, ViralConjunctivitis, Bacterial Conjunctivitis, Chlamydial & GonococcalConjunctivitis, Conjunctival Laceration, Episcleritis, Scleritis,Pingueculitis, Pterygium, Superior Limbic Keratoconjunctivitis (SLK ofTheodore), Toxic Conjunctivitis, Conjunctivitis with Pseudomembrane,Giant Papillary Conjunctivitis, Terrien's Marginal Degeneration,Acanthamoeba Keratitis, Fungal Keratitis, Filamentary Keratitis,Bacterial Keratitis, Keratitis Sicca/Dry Eye Syndrome, BacterialKeratitis, Herpes Simplex Keratitis, Sterile Corneal Infiltrates,Phlyctenulosis, Corneal Abrasion & Recurrent Corneal Erosion, CornealForeign Body, Chemical Burs, Epithelial Basement Membrane Dystrophy(EBMD), Thygeson's Superficial Punctate Keratopathy, Corneal Laceration,Salzmann's Nodular Degeneration, Fuchs' Endothelial Dystrophy,Crystalline Lens Subluxation, Ciliary-Block Glaucoma, Primary Open-AngleGlaucoma, Pigment Dispersion Syndrome and Pigmentary Glaucoma,Pseudoexfoliation Syndrom and Pseudoexfoliative Glaucoma, AnteriorUveitis, Primary Open Angle Glaucoma, Uveitic Glaucoma &Glaucomatocyclitic Crisis, Pigment Dispersion Syndrome & PigmentaryGlaucoma, Acute Angle Closure Glaucoma, Anterior Uveitis, Hyphema, AngleRecession Glaucoma, Lens Induced Glaucoma, Pseudoexfoliation Syndromeand Pseudoexfoliative Glaucoma, Axenfeld-Rieger Syndrome, NeovascularGlaucoma, Pars Planitis, Choroidal Rupture, Duane's Retraction Syndrome,Toxic/Nutritional Optic Neuropathy, Aberrant Regeneration of CranialNerve III, Intracranial Mass Lesions, Carotid-Cavernous Sinus Fistula,Anterior Ischemic Optic Neuropathy, Optic Disc Edema & Papilledema,Cranial Nerve III Palsy, Cranial Nerve IV Palsy, Cranial Nerve VI Palsy,Cranial Nerve VII (Facial Nerve) Palsy, Homer's Syndrome, InternuclearOphthalmoplegia, Optic Nerve Head Hypoplasia, Optic Pit, Tonic Pupil,Optic Nerve Head Drusen, Demyelinating Optic Neuropathy (Optic Neuritis,Retrobulbar Optic Neuritis), Amaurosis Fugax and Transient IschemicAttack, Pseudotumor Cerebri, Pituitary Adenoma, Molluscum Contagiosum,Canaliculitis, Verruca and Papilloma, Pediculosis and Pthiriasis,Blepharitis, Hordeolum, Preseptal Cellulitis, Chalazion, Basal CellCarcinoma, Herpes Zoster Ophthalmicus, Pediculosis & Phthiriasis,Blow-out Fracture, Chronic Epiphora, Dacryocystitis, Herpes. SimplexBlepharitis, Orbital Cellulitis, Senile Entropion, and Squamous CellCarcinoma.

By “dermatological disease” is mean any disease or condition of theskin, dermis, or any substructure therein such as hair, follicle, etc.Dermatological diseases, disorders, conditions, and traits can includepsoriasis, ectopic dermatitis, skin cancers such as melanoma and basalcell carcinoma, hair loss, hair removal, alterations in pigmentation,and any other disease, condition, or trait associated with the skin,dermis, or structures therein.

By “auditory disease” is mean any disease or condition of the auditorysystem, including the ear, such as the inner ear, middle ear, outer ear,auditory nerve, and any substructures therein. Auditory diseases,disorders, conditions, and traits can include hearing loss, deafness,tinnitus, Meniere's Disease, vertigo, balance and motion disorders, andany other disease, condition, or trait associated with the ear, orstructures therein.

By “metabolic disease” is meant any disease or condition affectingmetabolic pathways as in known in the art. Metabolic disease can resultin an abnormal metabolic process, either congenital due to inheritedenzyme abnormality (inborn errors of metabolism) or acquired due todisease of an endocrine organ or failure of a metabolically importantorgan such as the liver. In one embodiment, metabolic disease includeshyperlipidemia, hypercholesterolemia, cardiovascular disease,atherosclerosis, hypertension, diabetis (e.g., type I and/or type IIdiabetis), insulin resistance, and/or obesity.

By “cardiovascular disease” is meant and disease or condition affectingthe heart and vasculature, inlcuding but not limited to, coronary heartdisease (CHD), cerebrovascular disease (CVD), aortic stenosis,peripheral vascular disease, atherosclerosis, arteriosclerosis,myocardial infarction (heart attack), cerebrovascular diseases (stroke),transient ischaemic attacks (TIA), angina (stable and unstable), atrialfibrillation, arrhythmia, vavular disease, congestive heart failure,hypercholoesterolemia, type I hyperlipoproteinemia, type IIhyperlipoproteinemia, type III hyperlipoproteinemia, type IVhyperlipoproteinemia, type V hyperlipoproteinemia, secondaryhypertrigliceridemia, and familial lecithin cholesterol acyltransferasedeficiency.

In one embodiment of the present invention, each sequence of a siNAmolecule of the invention is independently about 15 to about 30nucleotides in length, in specific embodiments about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. Inanother embodiment, the siNA duplexes of the invention independentlycomprise about 15 to about 30 base pairs (e.g., about 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30). In anotherembodiment, one or more strands of the siNA molecule of the inventionindependently comprises about 15 to about 30 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) thatare complementary to a target nucleic acid molecule. In yet anotherembodiment, siNA molecules of the invention comprising hairpin orcircular structures are about 35 to about 55 (e.g., about 35, 40, 45, 50or 55) nucleotides in length, or about 38 to about 44 (e.g., about 38,39, 40, 41, 42, 43, or 44) nucleotides in length and comprising about 15to about 25 (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)base pairs. Exemplary siNA molecules of the invention are shown in TableII and/or FIGS. 4-5.

As used herein “cell” is used in its usual biological sense, and doesnot refer to an entire multicellular organism, e.g., specifically doesnot refer to a human. The cell can be present in an organism, e.g.,birds, plants and mammals such as humans, cows, sheep, apes, monkeys,swine, dogs, and cats. The cell can be prokaryotic (e.g., bacterialcell) or eukaryotic (e.g., mammalian or plant cell). The cell can be ofsomatic or germ line origin, totipotent or pluripotent, dividing ornon-dividing. The cell can also be derived from or can comprise a gameteor embryo, a stem cell, or a fully differentiated cell. The cell can bean isolated cell, purified cell, or substantially purified cell as isgenerally recognized in the art.

The siNA molecules of the invention are added directly, or can becomplexed with cationic lipids, packaged within liposomes, or otherwisedelivered to target cells or tissues. The nucleic acid or nucleic acidcomplexes can be locally administered to relevant tissues ex vivo, or invivo through local delivery to the lung, with or without theirincorporation in biopolymers. In particular embodiments, the nucleicacid molecules of the invention comprise sequences shown in Table IIand/or FIGS. 4-5. Examples of such nucleic acid molecules consistessentially of sequences defined in these tables and figures.Furthermore, the chemically modified constructs described in Table I andthe lipid nanoparticle (LNP) formulations shown in Table IV can beapplied to any siNA sequence or group of siNA sequences of theinvention.

In another aspect, the invention provides mammalian cells containing oneor more siNA molecules of this invention. The one or more siNA moleculescan independently be targeted to the same or different sites within atarget polynucleotide of the invention.

By “RNA” is meant a molecule comprising at least one ribonucleotideresidue. By “ribonucleotide” is meant a nucleotide with a hydroxyl groupat the 2′ position of a B-D-ribofuranose moiety. The terms includedouble-stranded RNA, single-stranded RNA, isolated RNA such as partiallypurified RNA, essentially pure RNA, synthetic RNA, recombinantlyproduced RNA, as well as altered RNA that differs from naturallyoccurring RNA by the addition, deletion, substitution and/or alterationof one or more nucleotides. Such alterations can include addition ofnon-nucleotide material, such as to the end(s) of the siNA orinternally, for example at one or more nucleotides of the RNA.Nucleotides in the RNA molecules of the instant invention can alsocomprise non-standard nucleotides, such as non-naturally occurringnucleotides or chemically synthesized nucleotides or deoxynucleotides.These altered RNAs can be referred to as analogs or analogs ofnaturally-occurring RNA.

By “subject” is meant an organism, which is a donor or recipient ofexplanted cells or the cells themselves. “Subject” also refers to anorganism to which the nucleic acid molecules of the invention can beadministered. A subject can be a mammal or mammalian cells, including ahuman or human cells. In one embodiment, the subject is an infant (e.g.,subjects that are less than 1 month old, or 1, 2, 3, 4, 5, 6, 7, 8, 910, 11, or 12 months old). In one embodiment, the subject is a toddler(e.g., 1, 2, 3, 4, 5 or 6 years old). In one embodiment, the subject isa senior (e.g., anyone over the age of about 65 years of age).

By “chemical modification” as used herein is meant any modification ofchemical structure of the nucleotides that differs from nucleotides ofnative siRNA or RNA. The term “chemical modification” encompasses theaddition, substitution, or modification of native siRNA or RNAnucleosides and nucleotides with modified nucleosides and modifiednucleotides as described herein or as is otherwise known in the art.Non-limiting examples of such chemical modifications include withoutlimitation compositions having any of Formulae I, II, III, IV, V, VI, orVII herein, phosphorothioate internucleotide linkages,2′-deoxyribonucleotides, 2′-O-methyl ribonucleotides, 2′-deoxy-2′-fluororibonucleotides, 4′-thio ribonucleotides, 2′-O-trifluoromethylnucleotides, 2′-O-ethyl-trifluoromethoxy nucleotides,2′-O-difluoromethoxy-ethoxy nucleotides (see for example U.S. Ser. No.10/981,966 filed Nov. 5, 2004, incorporated by reference herein), FANA,“universal base” nucleotides, “acyclic” nucleotides, 5-C-methylnucleotides, terminal glyceiyl and/or inverted deoxy abasic residueincorporation, or a modification having any of Formulae I-VII herein. Inone embodiment, the nucleic acid molecules of the invention (e.g, dsRNA,siNA etc.) are partially modified (e.g., about 5%, 10,%, 15%, 20%, 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%modified) with chemical modifications. In another embodiment, thenucleic acid molecules of the invention (e.g, dsRNA, siNA etc.) arecompletely modified (e.g., about 100% modified) with chemicalmodifications.

The term “phosphorothioate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise a sulfur atom.Hence, the term phosphorothioate refers to both phosphorothioate andphosphorodithioate intemucleotide linkages.

The term “phosphonoacetate” as used herein refers to an internucleotidelinkage having Formula I, wherein Z and/or W comprise an acetyl orprotected acetyl group.

The term “thiophosphonoacetate” as used herein refers to aninternucleotide linkage having Formula I, wherein Z comprises an acetylor protected acetyl group and W comprises a sulfur atom or alternately Wcomprises an acetyl or protected acetyl group and Z comprises a sulfuratom.

The term “universal base” as used herein refers to nucleotide baseanalogs that form base pairs with each of the natural DNA/RNA bases withlittle discrimination between them. Non-limiting examples of universalbases include C-phenyl, C-naphthyl and other aromatic derivatives,inosine, azole carboxamides, and nitroazole derivatives such as3-nitropyrrole, 4-nitroindole, 5-nitroindole, and 6-nitroindole as knownin the art (see for example Loakes, 2001, Nucleic Acids Research, 29,2437-2447).

The term “acyclic nucleotide” as used herein refers to any nucleotidehaving an cyclic ribose sugar, for example where any of the ribosecarbons (C1, C2, C3, C4, or C5), are independently or in combinationabsent from the nucleotide.

The nucleic acid molecules of the instant invention, individually, or incombination or in conjunction with other drugs, can be used to forpreventing or treating diseases, disorders, conditions, and traitsdescribed herein or otherwise known in the art, in a subject ororganism.

In one embodiment, the siNA molecules of the invention can beadministered to a subject or can be administered to other appropriatecells evident to those skilled in the art, individually or incombination with one or more drugs under conditions suitable for thetreatment.

In a further embodiment, the siNA molecules can be used in combinationwith other known treatments to prevent or treat diseases, disorders, orconditions in a subject or organism. For example, the describedmolecules could be used in combination with one or more known compounds,treatments, or procedures to prevent or treat diseases, disorders,conditions, and traits described herein in a subject or organism as areknown in the art.

In one embodiment, the invention features an expression vectorcomprising a nucleic acid sequence encoding at least one siNA moleculeof the invention, in a manner which allows expression of the siNAmolecule. For example, the vector can contain sequence(s) encoding bothstrands of a siNA molecule comprising a duplex. The vector can alsocontain sequence(s) encoding a single nucleic acid molecule that isself-complementary and thus forms a siNA molecule. Non-limiting examplesof such expression vectors are described in Paul et al, 2002, NatureBiotechnology, 19, 505; Miyagishi and Taira, 2002, Nature Biotechnology,19, 497; Lee et al., 2002, Nature Biotechnology, 19, 500; and Novina etal., 2002, Nature Medicine, advance online publicationdoi:10.1038/nm725.

In another embodiment, the invention features a mammalian cell, forexample, a human cell, including an expression vector of the invention.

In yet another embodiment, the expression vector of the inventioncomprises a sequence for a siNA molecule having complementarity to a RNAmolecule referred to by a Genbank Accession numbers, for example GenbankAccession Nos. described herein or in U.S. Provisional PatentApplication No. 60/363,124, U.S. Ser. No. 10/923,536 and/orPCT/US03/05028.

In one embodiment, an expression vector of the invention comprises anucleic acid sequence encoding two or more siNA molecules, which can bethe same or different.

In another aspect of the invention, siNA molecules that interact withtarget RNA molecules and down-regulate gene encoding target RNAmolecules (for example target RNA molecules referred to by GenbankAccession numbers herein) are expressed from transcription unitsinserted into DNA or RNA vectors. The recombinant vectors can be DNAplasmids or viral vectors. siNA expressing viral vectors can beconstructed based on, but not limited to, adeno-associated virus,retrovirus, adenovirus, or alphavirus. The recombinant vectors capableof expressing the siNA molecules can be delivered as described herein,and persist in target cells. Alternatively, viral vectors can be usedthat provide for transient expression of siNA molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecules bind and down-regulate gene function or expression via RNAinterference (RNAi). Delivery of siNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from a subject followed byreintroduction into the subject, or by any other means that would allowfor introduction into the desired target cell.

By “vectors” is meant any nucleic acid- and/or viral-based techniqueused to deliver a desired nucleic acid.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof, and from theclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a non-limiting example of a scheme for the synthesis ofsiNA molecules. The complementary siNA sequence strands, strand 1 andstrand 2, are synthesized in tandem and are connected by a cleavablelinkage, such as a nucleotide succinate or abasic succinate, which canbe the same or different from the cleavable linker used for solid phasesynthesis on a solid support. The synthesis can be either solid phase orsolution phase, in the example shown, the synthesis is a solid phasesynthesis. The synthesis is performed such that a protecting group, suchas a dimethoxytrityl group, remains intact on the terminal nucleotide ofthe tandem oligonucleotide. Upon cleavage and deprotection of theoligonucleotide, the two siNA strands spontaneously hybridize to form asiNA duplex, which allows the purification of the duplex by utilizingthe properties of the terminal protecting group, for example by applyinga trityl on purification method wherein only duplexes/oligonucleotideswith the terminal protecting group are isolated.

FIG. 2 shows a MALDI-TOF mass spectrum of a purified siNA duplexsynthesized by a method of the invention. The two peaks shown correspondto the predicted mass of the separate siNA sequence strands. This resultdemonstrates that the siNA duplex generated from tandem synthesis can bepurified as a single entity using a simple trityl-on purificationmethodology.

FIG. 3 shows a non-limiting proposed mechanistic representation oftarget RNA degradation involved in RNAi. Double-stranded RNA (dsRNA),which is generated by RNA-dependent RNA polymerase (RdRP) from foreignsingle-stranded RNA, for example viral, transposon, or other exogenousRNA, activates the DICER enzyme that in turn generates siNA duplexes.Alternately, synthetic or expressed siNA can be introduced directly intoa cell by appropriate means. An active siNA complex forms whichrecognizes a target RNA, resulting in degradation of the target RNA bythe RISC endonuclease complex or in the synthesis of additional RNA byRNA-dependent RNA polymerase (RdRP), which can activate DICER and resultin additional siNA molecules, thereby amplifying the RNAi response.

FIG. 4 shows non-limiting examples of chemically-modified siNAconstructs of the present invention. In the figure, N stands for anynucleotide (adenosine, guanosine, cytosine, uridine, or optionallythymidine, for example thymidine can be substituted in the overhangingregions designated by parenthesis (N N). Various modifications are shownfor the sense and antisense strands of the siNA constructs. The (N N)nucleotide positions can be chemically modified as described herein(e.g., 2′-O-methyl, 2′-deoxy-2′-fluoro etc.) and can be either derivedfrom a corresponding target nucleic acid sequence or not (see forexample FIG. 6C). Furthermore, the sequences shown in FIG. 4 canoptionally include a ribonucleotide at the 9^(th) position from the5′-end of the sense strand or the 11^(th) position based on the 5′-endof the guide strand by counting 11 nucleotide positions in from the5′-terminus of the guide strand (see FIG. 6C).

In FIG. 4, A shows that the sense strand comprises 21 nucleotideswherein the two terminal 3′-nucleotides are optionally base paired andwherein all nucleotides present are ribonucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety wherein the two terminal 3′-nucleotides areoptionally complementary to the target RNA sequence, and wherein allnucleotides present are ribonucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.

In FIG. 4, B shows that the sense strand comprises 21 nucleotideswherein the two terminal 3′-nucleotides are optionally base paired andwherein all pyrimidine nucleotides that may be present are2′deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2′-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the sense and antisensestrand.

In FIG. 4, C shows that the sense strand comprises 21 nucleotides having5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotidesare optionally base paired and wherein all pyrimidine nucleotides thatmay be present are 2′-O-methyl or 2′-deoxy-2′-fluoro modifiednucleotides except for (N N) nucleotides, which can compriseribonucleotides, deoxynucleotides, universal bases, or other chemicalmodifications described herein. The antisense strand comprises 21nucleotides, optionally having a 3′-terminal glyceryl moiety and whereinthe two terminal 3′-nucleotides are optionally complementary to thetarget RNA sequence, and wherein all pyrimidine nucleotides that may bepresent are 2′-deoxy-2′-fluoro modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified intemucleotide linkage as describedherein, shown as “s”, optionally connects the (N N) nucleotides in theantisense strand.

In FIG. 4, D shows that the sense strand comprises 21 nucleotides having5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotidesare optionally base paired and wherein all pyrimidine nucleotides thatmay be present are 2′-deoxy-2′-fluoro modified nucleotides except for (NN) nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,wherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-O-methyl modified nucleotides except for (N N)nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Amodified internucleotide linkage, such as a phosphorothioate,phosphorodithioate or other modified internucleotide linkage asdescribed herein, shown as “s”, optionally connects the (N N)nucleotides in the antisense strand.

In FIG. 4, E shows that the sense strand comprises 21 nucleotides having5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotidesare optionally base paired and wherein all pyrimidine nucleotides thatmay be present are 2′-deoxy-2′-fluoro modified nucleotides except for (NN) nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein. Theantisense strand comprises 21 nucleotides, optionally having a3′-terminal glyceryl moiety and wherein the two terminal 3′-nucleotidesare optionally complementary to the target RNA sequence, and wherein allpyrimidine nucleotides that may be present are 2′-deoxy-2-fluoromodified nucleotides and all purine nucleotides that may be present are2′-O-methyl modified nucleotides except for (N N) nucleotides, which cancomprise ribonucleotides, deoxynucleotides, universal bases, or otherchemical modifications described herein. A modified internucleotidelinkage, such as a phosphorothioate, phosphorodithioate or othermodified internucleotide linkage as described herein, shown as “s”,optionally connects the (N N) nucleotides in the antisense strand.

In FIG. 4, F shows that the sense strand comprises 21 nucleotides having5′- and 3′-terminal cap moieties wherein the two terminal 3′-nucleotidesare optionally base paired and wherein all pyrimidine nucleotides thatmay be present are 2′-deoxy-2′-fluoro modified nucleotides except for (NN) nucleotides, which can comprise ribonucleotides, deoxynucleotides,universal bases, or other chemical modifications described herein andwherein and all purine nucleotides that may be present are 2′-deoxynucleotides. The antisense strand comprises 21 nucleotides, optionallyhaving a 3′-terminal glyceryl moiety and wherein the two terminal3′-nucleotides are optionally complementary to the target RNA sequence,and having one 3′-terminal phosphorothioate internucleotide linkage andwherein all pyrimidine nucleotides that may be present are2′-deoxy-2′-fluoro modified nucleotides and all purine nucleotides thatmay be present are 2′-deoxy nucleotides except for (N N) nucleotides,which can comprise ribonucleotides, deoxynucleotides, universal bases,or other chemical modifications described herein. A modifiedinternucleotide linkage, such as a phosphorothioate, phosphorodithioateor other modified internucleotide linkage as described herein, shown as“s”, optionally connects the (N N) nucleotides in the antisense strand.The antisense strand of constructs A-F comprise sequence complementaryto any target nucleic acid sequence of the invention. Furthermore, whena glyceryl moiety (L) is present at the 3′-end of the antisense strandfor any construct shown in A-F of FIG. 4, the modified intemucleotidelinkage is optional.

FIG. 5 shows non-limiting examples of specific chemically-modified siNAsequences of the invention. A-F in FIG. 5 apply the chemicalmodifications described in A-F of FIG. 4 to an exemplary siNA sequence.Such chemical modifications can be applied to any siNA sequence for anytarget. Furthermore, the sequences shown in FIG. 5 can optionallyinclude a ribonucleotide at the 9th position from the 5′-end of thesense strand or the 11^(th) position based on the 5′-end of the guidestrand by counting 11 nucleotide positions in from the 5′-terminus ofthe guide strand (see FIG. 6C). In addition, the sequences shown in FIG.5 can optionally include terminal ribonucleotides at up to about 4positions at the 5′-end of the antisense strand (e.g., about 1, 2, 3, or4 terminal ribonucleotides at the 5′-end of the antisense strand).

FIG. 6A-C shows non-limiting examples of different siNA constructs ofthe invention.

The examples shown in FIG. 6A (constructs 1, 2, and 3) have 19representative base pairs; however, different embodiments of theinvention include any number of base pairs described herein. Bracketedregions represent nucleotide overhangs, for example, comprising about 1,2, 3, or 4 nucleotides in length, preferably about 2 nucleotides.Constructs 1 and 2 can be used independently for RNAi activity.Construct 2 can comprise a polynucleotide or non-nucleotide linker,which can optionally be designed as a biodegradable linker. In oneembodiment, the loop structure shown in construct 2 can comprise abiodegradable linker that results in the formation of construct 1 invivo and/or in vitro. In another example, construct 3 can be used togenerate construct 2 under the same principle wherein a linker is usedto generate the active siNA construct 2 in vivo and/or in vitro, whichcan optionally utilize another biodegradable linker to generate theactive siNA construct 1 in vivo and/or in vitro. As such, the stabilityand/or activity of the siNA constructs can be modulated based on thedesign of the siNA construct for use in vivo or in vitro and/or invitro.

The examples shown in FIG. 6B represent different variations of doublestranded nucleic acid molecule of the invention, such as microRNA, thatcan include overhangs, bulges, loops, and stein-loops resulting frompartial complementarily. Such motifs having bulges, loops, andstem-loops are generally characteristics of miRNA. The bulges, loops,and stem-loops can result from any degree of partial complementarily,such as mismatches or bulges of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore nucleotides in one or both strands of the double stranded nucleicacid molecule of the invention.

The example shown in FIG. 6C represents a model double stranded nucleicacid molecule of the invention comprising a 19 base pair duplex of two21 nucleotide sequences having dinucleotide 3′-overhangs. The top strand(1) represents the sense strand (passenger strand), the middle strand(2) represents the antisense (guide strand), and the lower strand (3)represents a target polynucleotide sequence. The dinucleotide overhangs(NN) can comprise sequence derived from the target polynucleotide. Forexample, the 3′-(NN) sequence in the guide strand can be complementaryto the 5′-[NN] sequence of the target polynucleotide. In addition, the5′-(NN) sequence of the passenger strand can comprise the same sequenceas the 5′-[NN] sequence of the target polynucleotide sequence. In otherembodiments, the overhangs (NN) are not derived from the targetpolynucleotide sequence, for example where the 3′-(NN) sequence in theguide strand are not complementary to the 5′-[NN] sequence of the targetpolynucleotide and the 5′-(NN) sequence of the passenger strand cancomprise different sequence from the 5′-[NN] sequence of the targetpolynucleotide sequence. In additional embodiments, any (NN) nucleotidesare chemically modified, e.g., as 2′-O-methyl, 2′-deoxy-2′-fluoro,and/or other modifications herein. Furthermore, the passenger strand cancomprise a ribonucleotide position N of the passenger strand. For therepresentative 19 base pair 21 mer duplex shown, position N can be 9nucleotides in from the 3′ end of the passenger strand. However, induplexes of differing length, the position N is determined based on the5′-end of the guide strand by counting 11 nucleotide positions in fromthe 5′-terminus of the guide strand and picking the corresponding basepaired nucleotide in the passenger strand. Cleavage by Ago2 takes placebetween positions 10 and 11 as indicated by the arrow. In additionalembodiments, there are two ribonucleotides, NN, at positions 10 and 11based on the 5′-end of the guide strand by counting 10 and 11 nucleotidepositions in from the 5′-terminus of the guide strand and picking thecorresponding base paired nucleotides in the passenger strand.

FIG. 7 is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate siNA hairpin constructs.

In FIG. 7, A shows that a DNA oligomer is synthesized with a5′-restriction site (R1) sequence followed by a region having sequenceidentical (sense region of siNA) to a predetermined target sequence,wherein the sense region comprises, for example, about 19, 20, 21, or 22nucleotides (N) in length, which is followed by a loop sequence ofdefined sequence (X), comprising, for example, about 3 to about 10nucleotides.

In FIG. 7, B shows that the synthetic construct is then extended by DNApolymerase to generate a hairpin structure having self-complementarysequence that will result in a siNA transcript having specificity for atarget sequence and having self-complementary sense and antisenseregions.

In FIG. 7, C shows that the construct is heated (for example to about95° C.) to linearize the sequence, thus allowing extension of acomplementary second DNA strand using a primer to the 3′-restrictionsequence of the first strand. The double-stranded DNA is then insertedinto an appropriate vector for expression in cells. The construct can bedesigned such that a 3′-terminal nucleotide overhang results from thetranscription, for example, by engineering restriction sites and/orutilizing a poly-U termination region as described in Paul et al., 2002,Nature Biotechnology, 29, 505-508.

FIG. 8 is a diagrammatic representation of a scheme utilized ingenerating an expression cassette to generate double-stranded siNAconstructs.

In FIG. 8, A shows that a DNA oligomer is synthesized with a5′-restriction (R1) site sequence followed by a region having sequenceidentical (sense region of siNA) to a predetermined target sequence,wherein the sense region comprises, for example, about 19, 20, 21, or 22nucleotides (N) in length, and which is followed by a 3′-restrictionsite (R2) which is adjacent to a loop sequence of defined sequence (X).

In FIG. 8, B shows that the synthetic construct is then extended by DNApolymerase to generate a hairpin structure having self-complementarysequence.

In FIG. 8, C shows that the construct is processed by restrictionenzymes specific to R1 and R2 to generate a double-stranded DNA which isthen inserted into an appropriate vector for expression in cells. Thetranscription cassette is designed such that a U6 promoter region flankseach side of the dsDNA which generates the separate sense and antisensestrands of the siNA. Poly T termination sequences can be added to theconstructs to generate U overhangs in the resulting transcript.

FIG. 9 is a diagrammatic representation of a method used to determinetarget sites for siNA mediated RNAi within a particular target nucleicacid sequence, such as messenger RNA.

In FIG. 9, A shows that a pool of siNA oligonucleotides are synthesizedwherein the antisense region of the siNA constructs has complementarityto target sites across the target nucleic acid sequence, and wherein thesense region comprises sequence complementary to the antisense region ofthe siNA.

In FIGS. 9, B and C show that the sequences (B) are pooled and areinserted into vectors such that (C) transfection of a vector into cellsresults in the expression of the siNA.

In FIG. 9, D shows that cells are sorted based on phenotypic change thatis associated with modulation of the target nucleic acid sequence.

In FIG. 9, E shows that the siNA is isolated from the sorted cells andis sequenced to identify efficacious target sites within the targetnucleic acid sequence.

FIG. 10 shows non-limiting examples of different stabilizationchemistries (1-10) that can be used, for example, to stabilize the3′-end of siNA sequences of the invention, including (1) [3-3′]-inverteddeoxyribose; (2) deoxyribonucleotide; (3)[5′-3′]-3′-deoxyribonucleotide; (4) [5′-3′]-ribonucleotide; (5)[5′-3]-3′-O-methyl ribonucleotide; (6) 3′-glyceryl; (7)[3′-5′]-3′-deoxyribonucleotide; (8) [3′-3′]-deoxyribonucleotide; (9)[5′-2′]-deoxyribonucleotide; and (10) [5-3′]-dideoxyribonucleotides. Inaddition to modified and unmodified backbone chemistries indicated inthe figure, these chemistries can be combined with different backbonemodifications as described herein, for example, backbone modificationshaving Formula I. In addition, the 2′-deoxy nucleotide shown 5′ to theterminal modifications shown can be another modified or unmodifiednucleotide or non-nucleotide described herein, for example modificationshaving any of Formulae I-VII or any combination thereof.

FIG. 11 shows a non-limiting example of a strategy used to identifychemically modified siNA constructs of the invention that are nucleaseresistant while preserving the ability to mediate RNAi activity.Chemical modifications are introduced into the siNA construct based oneducated design parameters (e.g., introducing 2′-modifications, basemodifications, backbone modifications, terminal cap modifications etc).The modified construct in tested in an appropriate system (e.g., humanserum for nuclease resistance, shown, or an animal model for PK/deliveryparameters). In parallel, the siNA construct is tested for RNAiactivity, for example in a cell culture system such as a luciferasereporter assay). Lead siNA constructs are then identified which possessa particular characteristic while maintaining RNAi activity, and can befurther modified and assayed once again. This same approach can be usedto identify siNA-conjugate molecules with improved pharmacokineticprofiles, delivery, and RNAi activity.

FIG. 12 shows non-limiting examples of phosphorylated siNA molecules ofthe invention, including linear and duplex constructs and asymmetricderivatives thereof.

FIG. 13 shows non-limiting examples of chemically modified terminalphosphate groups of the invention.

FIG. 14A shows a non-limiting example of methodology used to design selfcomplementary DFO constructs utilizing palindrome and/or repeat nucleicacid sequences that are identified in a target nucleic acid sequence.(i) A palindrome or repeat sequence is identified in a nucleic acidtarget sequence. (ii) A sequence is designed that is complementary tothe target nucleic acid sequence and the palindrome sequence. (iii) Aninverse repeat sequence of the non-palindrome/repeat portion of thecomplementary sequence is appended to the 3′-end of the complementarysequence to generate a self complementary DFO molecule comprisingsequence complementary to the nucleic acid target. (iv) The DFO moleculecan self-assemble to form a double stranded oligonucleotide. FIG. 14Bshows a non-limiting representative example of a duplex formingoligonucleotide sequence. FIG. 14C shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence. FIG. 14D shows a non-limiting example of theself assembly schematic of a representative duplex formingoligonucleotide sequence followed by interaction with a target nucleicacid sequence resulting in modulation of gene expression.

FIG. 15 shows a non-limiting example of the design of self complementaryDFO constructs utilizing palindrome and/or repeat nucleic acid sequencesthat are incorporated into the DFO constructs that have sequencecomplementary to any target nucleic acid sequence of interest.Incorporation of these palindrome/repeat sequences allow the design ofDFO constructs that form duplexes in which each strand is capable ofmediating modulation of target gene expression, for example by RNAi.First, the target sequence is identified. A complementary sequence isthen generated in which nucleotide or non-nucleotide modifications(shown as X or Y) are introduced into the complementary sequence thatgenerate an artificial palindrome (shown as XYXYXY in the Figure). Aninverse repeat of the non-palindrome/repeat complementary sequence isappended to the 3′-end of the complementary sequence to generate a selfcomplementary DFO comprising sequence complementary to the nucleic acidtarget. The DFO can self-assemble to form a double strandedoligonucleotide.

FIG. 16 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences. A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the first andsecond complementary regions are situated at the 3′-ends of eachpolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. B shows a non-limiting example of a multifunctional siNAmolecule having a first region that is complementary to a first targetnucleic acid sequence (complementary region 1) and a second region thatis complementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 5′-ends of each polynucleotide sequence in themultifunctional siNA. The dashed portions of each polynucleotidesequence of the multifunctional siNA construct have complementarity withregard to corresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 17 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences. A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe second complementary region is situated at the 3′-end of thepolynucleotide sequence in the multifunctional siNA. The dashed portionsof each polynucleotide sequence of the multifunctional siNA constructhave complementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. B shows a non-limiting example of a multifunctional siNAmolecule having a first region that is complementary to a first targetnucleic acid sequence (complementary region 1) and a second region thatis complementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first complementary region is situated at the5′-end of the polynucleotide sequence in the multifunctional siNA. Thedashed portions of each polynucleotide sequence of the multifunctionalsiNA construct have complementarity with regard to correspondingportions of the siNA duplex, but do not have complementarity to thetarget nucleic acid sequences. In one embodiment, these multifunctionalsiNA constructs are processed in vivo or in vitro to generatemultifunctional siNA constructs as shown in FIG. 16.

FIG. 18 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising two separate polynucleotide sequences that areeach capable of mediating RNAi directed cleavage of differing targetnucleic acid sequences and wherein the multifunctional siNA constructfurther comprises a self complementary, palindrome, or repeat region,thus enabling shorter bifunctional siNA constructs that can mediate RNAinterference against differing target nucleic acid sequences. A shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first and second complementary regions are situated at the 3′-endsof each polynucleotide sequence in the multifunctional siNA, and whereinthe first and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarity with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. B shows a non-limiting example of a multifunctional siNAmolecule having a first region that is complementary to a first targetnucleic acid sequence (complementary region 1) and a second region thatis complementary to a second target nucleic acid sequence (complementaryregion 2), wherein the first and second complementary regions aresituated at the 5′-ends of each polynucleotide sequence in themultifunctional siNA, and wherein the first and second complementaryregions further comprise a self complementary, palindrome, or repeatregion. The dashed portions of each polynucleotide sequence of themultifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences.

FIG. 19 shows non-limiting examples of multifunctional siNA molecules ofthe invention comprising a single polynucleotide sequence comprisingdistinct regions that are each capable of mediating RNAi directedcleavage of differing target nucleic acid sequences and wherein themultifunctional siNA construct further comprises a self complementary,palindrome, or repeat region, thus enabling shorter bifunctional siNAconstructs that can mediate RNA interference against differing targetnucleic acid sequences. A shows a non-limiting example of amultifunctional siNA molecule having a first region that iscomplementary to a first target nucleic acid sequence (complementaryregion 1) and a second region that is complementary to a second targetnucleic acid sequence (complementary region 2), wherein the secondcomplementary region is situated at the 3′-end of the polynucleotidesequence in the multifunctional siNA, and wherein the first and secondcomplementary regions further comprise a self complementary, palindrome,or repeat region. The dashed portions of each polynucleotide sequence ofthe multifunctional siNA construct have complementarity with regard tocorresponding portions of the siNA duplex, but do not havecomplementarity to the target nucleic acid sequences. B shows anon-limiting example of a multifunctional siNA molecule having a firstregion that is complementary to a first target nucleic acid sequence(complementary region 1) and a second region that is complementary to asecond target nucleic acid sequence (complementary region 2), whereinthe first complementary region is situated at the 5′-end of thepolynucleotide sequence in the multifunctional siNA, and wherein thefirst and second complementary regions further comprise a selfcomplementary, palindrome, or repeat region. The dashed portions of eachpolynucleotide sequence of the multifunctional siNA construct havecomplementarily with regard to corresponding portions of the siNAduplex, but do not have complementarity to the target nucleic acidsequences. In one embodiment, these multifunctional siNA constructs areprocessed in vivo or in vitro to generate multifunctional siNAconstructs as shown in FIG. 18.

FIG. 20 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidmolecules, such as separate RNA molecules encoding differing proteins(e.g., any of targets herein), for example, a cytokine and itscorresponding receptor, differing viral strains, a virus and a cellularprotein involved in viral infection or replication, or differingproteins involved in a common or divergent biologic pathway that isimplicated in the maintenance of progression of disease. Each strand ofthe multifunctional siNA construct comprises a region havingcomplementarity to separate target nucleic acid molecules. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target. These design parameterscan include destabilization of each end of the siNA construct (see forexample Schwarz et al., 2003, Cell, 115, 199-208). Such destabilizationcan be accomplished for example by using guanosine-cytidine base pairs,alternate base pairs (e.g., wobbles), or destabilizing chemicallymodified nucleotides at terminal nucleotide positions as is known in theart.

FIG. 21 shows a non-limiting example of how multifunctional siNAmolecules of the invention can target two separate target nucleic acidsequences within the same target nucleic acid molecule, such asalternate coding regions of a RNA, coding and non-coding regions of aRNA, or alternate splice variant regions of a RNA. Each strand of themultifunctional siNA construct comprises a region having complementarilyto the separate regions of the target nucleic acid molecule. Themultifunctional siNA molecule is designed such that each strand of thesiNA can be utilized by the RISC complex to initiate RNA interferencemediated cleavage of its corresponding target region. These designparameters can include destabilization of each end of the siNA construct(see for example Schwarz et al., 2003, Cell, 115, 199-208). Suchdestabilization can be accomplished for example by usingguanosine-cytidine base pairs, alternate base pairs (e.g., wobbles), ordestabilizing chemically modified nucleotides at terminal nucleotidepositions as is known in the art.

FIG. 22 shows non-limiting examples of tethered multifunctional siNAconstructs of the invention. In the examples shown, a linker (e.g.,nucleotide or non-nucleotide linker) connects two siNA regions (e.g.,two sense, two antisense, or alternately a sense and an antisense regiontogether. Separate sense (or sense and anti sense) sequencescorresponding to a first target sequence and second target sequence arehybridized to their corresponding sense and/or antisense sequences inthe multifunctional siNA. In addition, various conjugates, ligands,aptamers, polymers or reporter molecules can be attached to the linkerregion for selective or improved delivery and/or pharmacokineticproperties. A show that the multifunctional siNA is assembled from twoseparate double-stranded siNAs, with the 5′-end of one sense strand ofthe siNA is tethered to the 5′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, point away (in the opposite direction) from eachother. B shows that the multifunctional siNA is assembled from twoseparate double-stranded siNAs, with the 3′-end of one sense strand ofthe siNA is tethered to the 3′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, face each other. C and D show that the multifunctionalsiNA is assembled from two separate double-stranded siNAs, with the5′-end of one sense strand of the siNA is tethered to the 3′-end of thesense strand of the other siNA molecule, such that the 5′-end of the oneof the antisense siNA strands annealed to their corresponding sensestrand that are tethered to each other at one end, faces the 3′-end ofthe other antisense strand. E shows that the multifunctional siNA isassembled from two separate double-stranded siNAs, with the 5′-end ofone antisense strand of the siNA is tethered to the 5′-end of theantisense strand of the other siNA molecule, such that the 3′-end of theone of the sense siNA strands annealed to their corresponding antisensesense strand that are tethered to each other at one end, faces the3′-end of the other sense strand. F shows that the multifunctional siNAis assembled from two separate double-stranded siNAs, with the 3′-end ofone antisense strand of the siNA is tethered to the 3′-end of theantisense strand of the other siNA molecule, such that the 5′-end of theone of the sense siNA strands annealed to their corresponding antisensesense strand that are tethered to each other at one end, faces the3′-end of the other sense strand. G and H show that the multifunctionalsiNA is assembled from two separate double-stranded siNAs, with the5′-end of one antisense strand of the siNA is tethered to the 3′-end ofthe antisense strand of the other siNA molecule, such that the 5′-end ofthe one of the sense siNA strands annealed to their correspondingantisense sense strand that are tethered to each other at one end, facesthe 3′-end of the other sense strand.

FIG. 23 shows a non-limiting example of various dendrimer basedmultifunctional siNA designs.

FIG. 24 shows a non-limiting example of various supramolecularmultifunctional siNA designs.

FIG. 25 shows a non-limiting example of a dicer enabled multifunctionalsiNA design using a 30 nucleotide precursor siNA construct. A 30 basepair duplex is cleaved by Dicer into 22 and 8 base pair products fromeither end (8 b.p. fragments not shown). For ease of presentation theoverhangs generated by dicer are not shown—but can be compensated for.Three targeting sequences are shown. The required sequence identityoverlapped is indicated by grey boxes. The N's of the parent 30 b.p.siNA are suggested sites of 2′-OH positions to enable Dicer cleavage ifthis is tested in stabilized chemistries. Note that processing of a30mer duplex by Dicer RNase III does not give a precise 22+8 cleavage,but rather produces a series of closely related products (with 22+8being the primary site). Therefore, processing by Dicer will yield aseries of active siNAs.

FIG. 26 shows a non-limiting example of a dicer enabled multifunctionalsiNA design using a 40 nucleotide precursor siNA construct. A 40 basepair duplex is cleaved by Dicer into 20 base pair products from eitherend. For ease of presentation the overhangs generated by dicer are notshown—but can be compensated for. Four targeting sequences are shown.The target sequences having homology are enclosed by boxes. This designformat can be extended to larger RNAs. If chemically stabilized siNAsare bound by Dicer, then strategically located ribonucleotide linkagescan enable designer cleavage products that permit our more extensiverepertoire of multifunctional designs. For example cleavage products notlimited to the Dicer standard of approximately 22-nucleotides can allowmultifunctional siNA constructs with a target sequence identity overlapranging from, for example, about 3 to about 15 nucleotides.

FIG. 27 shows a non-limiting example of additional multifunctional siNAconstruct designs of the invention. In one example, a conjugate, ligand,aptamer, label, or other moiety is attached to a region of themultifunctional siNA to enable improved delivery or pharmacokineticprofiling.

FIG. 28 shows a non-limiting example of additional multifunctional siNAconstruct designs of the invention. In one example, a conjugate, ligand,aptamer, label, or other moiety is attached to a region of themultifunctional siNA to enable improved delivery or pharmacokineticprofiling.

FIG. 29 shows a non-limiting example of a cholesterol linkedphosphoramidite that can be used to synthesize cholesterol conjugatedsiNA molecules of the invention. An example is shown with thecholesterol moiety linked to the 5′-end of the sense strand of a siNAmolecule.

FIG. 30 shows a non-limiting example of inhibition of HBV S antigen(HBsAg) in vitro using various siNA constructs having selectmodification patterns that include ribonucleotides at select positionsand which target HBV site 262 RNA.

FIG. 31 shows a non-limiting example of inhibition of HBV S antigen(HBsAg) in vitro using various siNA constructs having selectmodification patterns that include ribonucleotides at select positionsand which target HBV site 263 RNA.

FIG. 32 shows a non-limiting example of inhibition of HBV S antigen(HBsAg) in vitro using various siNA constructs having selectmodification patterns that include ribonucleotides at select positionsand which target HBV site 1583 RNA.

FIG. 33 shows a non-limiting example of dose dependent inhibition of HBVS antigen (HBsAg) in vitro using two different siNA constructs havingselect modification patterns that include ribonucleotides at selectpositions and which target HBV site 1583 RNA.

FIG. 34 shows a non-limiting example of dose dependent inhibition of HBVS antigen (HBsAg) in vitro using two different siNA constructs havingselect modification patterns that include ribonucleotides at selectpositions and which target HBV site 1583 RNA.

FIG. 35 shows a non-limiting example of inhibition of HBV S antigen(HBsAg) in vitro using various siNA constructs having selectmodification patterns that include ribonucleotides at select positionsand which target HBV sites 262 and 263 RNA.

FIG. 36 shows a non-limiting example of dose dependent inhibition of HCVRNA expression in vitro using Stab 25 and Stab 29 siNA constructstargeting sites 327, 282, and 304 RNA.

FIG. 37 shows a non-limiting example of the in vivo inhibition of HBVDNA in mice using LNP-086 and LNP-061 formulated siNA molecules of theinvention with different overhang chemistries. Active LNP-086 andLNP-061 siNA constructs were evaluated compared to PBS control, andinverted control groups. As shown in the figure, siNA constructs with2′-O-methyl overhangs provide potent anti-HBV activity in this model.

FIG. 38 shows a non-limiting example HBV263M-LNP-086 mediated reductionin levels of serum HBV DNA in vivo in HBV-replicating mice that weretreated with doses of 0.3, 1, or 3 mg/kg/day for three days compared tocontrol siNA or PBS groups. Levels of serum HBV DNA were equivalent inthe control siNA and PBS treated groups, demonstrating the sequencespecificity of the anti-HBV activity, and the absence of non-specificlipid effects.

FIG. 39 shows a non-limiting example of HBV263M-LNP-086 mediatedreduction in levels of serum HBV HBsAg in vivo in HBV-replicating micethat were treated with doses of 0.3, 1, or 3 mg/kg/day for three dayscompared to control siNA or PBS groups. Levels of serum HBV HBsAg wereequivalent in the control siNA and PBS treated groups, demonstrating thesequence specificity of the anti-HBV activity, and the absence ofnon-specific lipid effects.

FIG. 40 shows a non-limiting example of the duration of siNA-mediatedreductions in HBV levels in a mouse model of HBV infection.HBV-replicating mice were treated with HBV263M-LNP-086 orHBV263Minv-LNP-086 at doses of 3 mg/kg/day for three days, followed byanalysis of HBV serum titers at days 3, 7, and 14 after the last dose.As shown in the figure, the anti-HBV activity was persistent, withsignificant activity still observed at day 7 (2.0 log 10 reduction) andday 14 (1.5 log 10 reduction).

FIG. 41 shows a non-limiting example of liver specific HBV RNA cleavagemediated by the active HBV263M-LNP-086 formulation in a mouse model ofHBV infection. Mice replicating HBV were treated with doses ofHBV263M-LNP-086 at 0.3, 1, 3, 10 mg/kg/day or the HBV263invM-LNP controlat 10 mg/kg for three days, and levels of liver HBV RNA were determined3 days following the last dose. Dose-dependent reduction of liver HBVRNA was observed, with decreases of 90%, 66.5%, 18%, and 4% seen in the10, 3, 1, and 0.3 mg/kg HBV263M-LNP treatment groups respectivelycompared to the HBV263invM-LNP-086 control at 10 mg/kg.

FIG. 42 shows a non-limiting example of the demonstration that thereduction in liver HBV RNA is due to RNAi-mediated cleavage of HBV RNA.5′ rapid amplification of cDNA ends (RACE) analysis was used to detectcleavage of the HBV RNA at the predicted site. HBV-replicating mice weretreated with HBV263M-LNP-086 or HBV263Minv-LNP-086 at a dose of 3mg/kg/d for 3 days. The animals were sacrificed at 3, 7, or 14 daysfollowing the last dose, and total liver RNA was isolated. Ligation ofan adaptor sequence to the free 5′ends of the RNA population, andsubsequent RT-PCR with adaptor and HBV specific primers was expected toresult in a PCR product of 145 by if the HBV RNA had been cleaved at thepredicted target site. As shown the figure, the expected amplificationproduct was observed in the HBV263 active siNA-treated samples at eachtime point, but not in the HBV263 control samples. PCR products werethen subcloned and sequenced, confirming the correct junction betweenthe adaptor sequence and the predicted cleavage site of the HBV263 siNA.This result establishes that the reduction in HBV RNA observed in theliver was due to specific RNAi-mediated cleavage of the HBV RNA in theliver. In addition, the detection of specific HBV RNA cleavage productsat the 7 and 14 day time points demonstrates that the duration of thesiNA activity against HBV is due to continued cleavage of HBV RNA.

FIG. 43 shows a non-limiting example of the pharmacokinetic propertiesof HBV263M-LNP-086 as determined in mice after a single 3 mg/kg dose. Ahybridization method was used to detect the HBV263M siNA in plasma andliver over time. HBV263M was eliminated rapidly in plasma with anelimination T1/2 of approximately 1.7 h. However, HBV263M was detectedin the liver throughout the 14 d sampling period and had an eliminationT1/2 of 4 days. A maximum concentration of 31.3±17.8 ng/mg(mean±standard deviation) was observed in the liver at 1 hour andcorresponded to 65±32% of the siNA dose. At 14 days, 1.4±0.7% of thedose remained intact in the liver. The prolonged siNA-mediated anti-HBVactivity observed in the mouse model correlates well with this extendedresidence time of the siNA in the liver.

DETAILED DESCRIPTION OF THE INVENTION

Mechanism of Action of Nucleic Acid Molecules of the Invention

The discussion that follows discusses the proposed mechanism of RNAinterference mediated by short interfering RNA as is presently known,and is not meant to be limiting and is not an admission of prior art.Applicant demonstrates herein that chemically-modified short interferingnucleic acids possess similar or improved capacity to mediate RNAi as dosiRNA molecules and are expected to possess improved stability andactivity in vivo; therefore, this discussion is not meant to be limitingonly to siRNA and can be applied to siNA as a whole. By “improvedcapacity to mediate RNAi” or “improved RNAi activity” is meant toinclude RNAi activity measured in vitro and/or in vivo where the RNAiactivity is a reflection of both the ability of the siNA to mediate RNAiand the stability of the siNAs of the invention. In this invention, theproduct of these activities can be increased in vitro and/or in vivocompared to an all RNA siRNA or a siNA containing a plurality ofribonucleotides. In some cases, the activity or stability of the siNAmolecule can be decreased (i.e., less than ten-fold), but the overallactivity of the siNA molecule is enhanced in vitro and/or in vivo.

RNA interference refers to the process of sequence specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire et al., 1998, Nature, 391, 806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes whichis commonly shared by diverse flora and phyla (Fire et al., 1999, Trendsgenet., 15, 358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or the random integration of transposonelements into a host genome via a cellular response that specificallydestroys homologous single-stranded RNA or viral genomic RNA. Thepresence of dsRNA in cells triggers the RNAi response though a mechanismthat has yet to be fully characterized. This mechanism appears to bedifferent from the interferon response that results from dsRNA-mediatedactivation of protein kinase PKR and 2′, 5′-oligoadenylate synthetaseresulting in non-specific cleavage of mRNA by ribonuclease L.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as Dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein et al., 2001, Nature, 409, 363).Short interfering RNAs derived from Dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes. Dicer has also been implicated in the excision of 21- and22-nucleotide small temporal RNAs (stRNAs) from precursor RNA ofconserved structure that are implicated in translational control(Hutvagner et al., 2001, Science, 293, 834). The RNAi response alsofeatures an endonuclease complex containing a siRNA, commonly referredto as an RNA-induced silencing complex (RISC), which mediates cleavageof single-stranded RNA having sequence homologous to the siRNA. Cleavageof the target RNA takes place in the middle of the region complementaryto the guide sequence of the siRNA duplex (Elbashir et al., 2001, genesDev., 15, 188). In addition, RNA interference can also involve small RNA(e.g., micro-RNA or miRNA) mediated gene silencing, presumably thoughcellular mechanisms that regulate chromatin structure and therebyprevent transcription of target gene sequences (see for exampleAllshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science,297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall etal., 2002, Science, 297, 2232-2237). As such, siNA molecules of theinvention can be used to mediate gene silencing via interaction with RNAtranscripts or alternately by interaction with particular genesequences, wherein such interaction results in gene silencing either atthe transcriptional level or post-transcriptional level.

RNAi has been studied in a variety of systems. Fire et al., 1998,Nature, 391, 806, were the first to observe RNAi in C. elegans. Wiannyand Goetz, 1999, Nature Cell Biol., 2, 70, describe RNAi mediated bydsRNA in mouse embryos. Hammond et al., 2000, Nature, 404, 293, describeRNAi in Drosophila cells transfected with dsRNA. Elbashir et al., 2001,Nature, 411, 494, describe RNAi induced by introduction of duplexes ofsynthetic 21-nucleotide RNAs in cultured mammalian cells including humanembryonic kidney and HeLa cells. Recent work in Drosophila embryoniclysates has revealed certain requirements for siRNA length, structure,chemical composition, and sequence that are essential to mediateefficient RNAi activity. These studies have shown that 21 nucleotidesiRNA duplexes are most active when containing two 2-nucleotide3′-terminal nucleotide overhangs. Furthermore, substitution of one orboth siRNA strands with 2′-deoxy or 2′-O-methyl nucleotides abolishesRNAi activity, whereas substitution of 3′-terminal siRNA nucleotideswith deoxy nucleotides was shown to be tolerated. Mismatch sequences inthe center of the siRNA duplex were also shown to abolish RNAi activity.In addition, these studies also indicate that the position of thecleavage site in the target RNA is defined by the 5′-end of the siRNAguide sequence rather than the 3′-end (Elbashir et al., 2001, EMBO J.,20, 6877). Other studies have indicated that a 5′-phosphate on thetarget-complementary strand of a siRNA duplex is required for siRNAactivity and that ATP is utilized to maintain the 5′-phosphate moiety onthe siRNA (Nykanen et al., 2001, Cell, 107, 309); however, siRNAmolecules lacking a 5′-phosphate are active when introduced exogenously,suggesting that 5′-phosphorylation of siRNA constructs may occur invivo.

Duplex Forming Oligonucleotides (DFO) of the Invention

In one embodiment, the invention features siNA molecules comprisingduplex forming oligonucleotides (DFO) that can self-assemble into doublestranded oligonucleotides. The duplex forming oligonucleotides of theinvention can be chemically synthesized or expressed from transcriptionunits and/or vectors. The DFO molecules of the instant invention provideuseful reagents and methods for a variety of therapeutic, diagnostic,agricultural, veterinary, target validation, genomic discovery, geneticengineering and pharmacogenomic applications.

Applicant demonstrates herein that certain oligonucleotides, refereed toherein for convenience but not limitation as duplex formingoligonucleotides or DFO molecules, are potent mediators of sequencespecific regulation of gene expression. The oligonucleotides of theinvention are distinct from other nucleic acid sequences known in theart (e.g, siRNA, miRNA, stRNA, shRNA, antisense oligonucleotides etc.)in that they represent a class of linear polynucleotide sequences thatare designed to self-assemble into double stranded oligonucleotides,where each strand in the double stranded oligonucleotides comprises anucleotide sequence that is complementary to a target nucleic acidmolecule. Nucleic acid molecules of the invention can thus self assembleinto functional duplexes in which each strand of the duplex comprisesthe same polynucleotide sequence and each strand comprises a nucleotidesequence that is complementary to a target nucleic acid molecule.

generally, double stranded oligonucleotides are formed by the assemblyof two distinct oligonucleotide sequences where the oligonucleotidesequence of one strand is complementary to the oligonucleotide sequenceof the second strand; such double stranded oligonucleotides areassembled from two separate oligonucleotides, or from a single moleculethat folds on itself to form a double stranded structure, often referredto in the field as hairpin stern-loop structure (e.g., shRNA or shorthairpin RNA). These double stranded oligonucleotides known in the artall have a common feature in that each strand of the duplex has adistinct nucleotide sequence.

Distinct from the double stranded nucleic acid molecules known in theart, the applicants have developed a novel, potentially cost effectiveand simplified method of forming a double stranded nucleic acid moleculestarting from a single stranded or linear oligonucleotide. The twostrands of the double stranded oligonucleotide formed according to theinstant invention have the same nucleotide sequence and are notcovalently linked to each other. Such double-stranded oligonucleotidesmolecules can be readily linked post-synthetically by methods andreagents known in the art and are within the scope of the invention. Inone embodiment, the single stranded oligonucleotide of the invention(the duplex forming oligonucleotide) that forms a double strandedoligonucleotide comprises a first region and a second region, where thesecond region includes a nucleotide sequence that is an inverted repeatof the nucleotide sequence in the first region, or a portion thereof,such that the single stranded oligonucleotide self assembles to form aduplex oligonucleotide in which the nucleotide sequence of one strand ofthe duplex is the same as the nucleotide sequence of the second strand.Non-limiting examples of such duplex forming oligonucleotides areillustrated in FIGS. 14 and 15. These duplex forming oligonucleotides(DFOs) can optionally include certain palindrome or repeat sequenceswhere such palindrome or repeat sequences are present in between thefirst region and the second region of the DFO.

In one embodiment, the invention features a duplex formingoligonucleotide (DFO) molecule, wherein the DFO comprises a duplexforming self complementary nucleic acid sequence that has nucleotidesequence complementary to a target nucleic acid sequence. The DFOmolecule can comprise a single self complementary sequence or a duplexresulting from assembly of such self complementary sequences.

In one embodiment, a duplex forming oligonucleotide (DFO) of theinvention comprises a first region and a second region, wherein thesecond region comprises a nucleotide sequence comprising an invertedrepeat of nucleotide sequence of the first region such that the DFOmolecule can assemble into a double stranded oligonucleotide. Suchdouble stranded oligonucleotides can act as a short interfering nucleicacid (siNA) to modulate gene expression. Each strand of the doublestranded oligonucleotide duplex formed by DFO molecules of the inventioncan comprise a nucleotide sequence region that is complementary to thesame nucleotide sequence in a target nucleic acid molecule (e.g., targetRNA).

In one embodiment, the invention features a single stranded DFO that canassemble into a double stranded oligonucleotide. The applicant hassurprisingly found that a single stranded oligonucleotide withnucleotide regions of self complementarity can readily assemble intoduplex oligonucleotide constructs. Such DFOs can assemble into duplexesthat can inhibit gene expression in a sequence specific manner. The DFOmolecules of the invention comprise a first region with nucleotidesequence that is complementary to the nucleotide sequence of a secondregion and where the sequence of the first region is complementary to atarget nucleic acid (e.g., RNA). The DFO can form a double strandedoligonucleotide wherein a portion of each strand of the double strandedoligonucleotide comprises a sequence complementary to a target nucleicacid sequence.

In one embodiment, the invention features a double strandedoligonucleotide, wherein the two strands of the double strandedoligonucleotide are not covalently linked to each other, and whereineach strand of the double stranded oligonucleotide comprises anucleotide sequence that is complementary to the same nucleotidesequence in a target nucleic acid molecule or a portion thereof (e.g.,target RNA target). In another embodiment, the two strands of the doublestranded oligonucleotide share an identical nucleotide sequence of atleast about 15, preferably at least about 16, 17, 18, 19, 20, or 21nucleotides.

In one embodiment, a DFO molecule of the invention comprises a structurehaving Formula DFO-I:

5′-p-X Z X′-3′wherein Z comprises a palindromic or repeat nucleic acid sequenceoptionally with one or more modified nucleotides (e.g., nucleotide witha modified base, such as 2-amino purine, 2-amino-1,6-dihydro purine or auniversal base), for example of length about 2 to about 24 nucleotidesin even numbers (e.g., about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, or 22or 24 nucleotides), X represents a nucleic acid sequence, for example oflength of about 1 to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides),X′ comprises a nucleic acid sequence, for example of length about 1 andabout 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotidesequence complementarity to sequence X or a portion thereof, p comprisesa terminal phosphate group that can be present or absent, and whereinsequence X and Z, either independently or together, comprise nucleotidesequence that is complementary to a target nucleic acid sequence or aportion thereof and is of length sufficient to interact (e.g., basepair) with the target nucleic acid sequence or a portion thereof (e.g.,target RNA target). For example, X independently can comprise a sequencefrom about 12 to about 21 or more (e.g., about 12, 13, 14, 15, 16, 17,18, 19, 20, 21, or more) nucleotides in length that is complementary tonucleotide sequence in a target RNA or a portion thereof. In anothernon-limiting example, the length of the nucleotide sequence of X and Ztogether, when X is present, that is complementary to the target or aportion thereof (e.g., target RNA target) is from about 12 to about 21or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,or more). In yet another non-limiting example, when X is absent, thelength of the nucleotide sequence of Z that is complementary to thetarget or a portion thereof is from about 12 to about 24 or morenucleotides (e.g., about 12, 14, 16, 18, 20, 22, 24, or more). In oneembodiment X, Z and X′ are independently oligonucleotides, where Xand/or Z comprises a nucleotide sequence of length sufficient tointeract (e.g., base pair) with a nucleotide sequence in the target or aportion thereof (e.g., target RNA target). In one embodiment, thelengths of oligonucleotides X and X′ are identical. In anotherembodiment, the lengths of oligonucleotides X and X′ are not identical.In another embodiment, the lengths of oligonucleotides X and Z, or Z andX′, or X, Z and X′ are either identical or different.

When a sequence is described in this specification as being of“sufficient” length to interact (i.e., base pair) with another sequence,it is meant that the length is such that the number of bonds (e.g.,hydrogen bonds) formed between the two sequences is enough to enable thetwo sequence to form a duplex under the conditions of interest. Suchconditions can be in vitro (e.g., for diagnostic or assay purposes) orin vivo (e.g., for therapeutic purposes). It is a simple and routinematter to determine such lengths.

In one embodiment, the invention features a double strandedoligonucleotide construct having Formula DFO-I(a):

5′-p-X Z X′-3′ 3′-X′ Z X-p-5′wherein Z comprises a palindromic or repeat nucleic acid sequence orpalindromic or repeat-like nucleic acid sequence with one or moremodified nucleotides (e.g., nucleotides with a modified base, such as2-amino purine, 2-amino-1,6-dihydro purine or a universal base), forexample of length about 2 to about 24 nucleotides in even numbers (e.g.,about 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 nucleotides), Xrepresents a nucleic acid sequence, for example of length about 1 toabout 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, or 21 nucleotides), X′ comprises anucleic acid sequence, for example of length about 1 to about 21nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 or 21 nucleotides) having nucleotide sequencecomplementarity to sequence X or a portion thereof, p comprises aterminal phosphate group that can be present or absent, and wherein eachX and Z independently comprises a nucleotide sequence that iscomplementary to a target nucleic acid sequence or a portion thereof(e.g., target RNA target) and is of length sufficient to interact withthe target nucleic acid sequence of a portion thereof (e.g., target RNAtarget). For example, sequence X independently can comprise a sequencefrom about 12 to about 21 or more nucleotides (e.g., about 12, 13, 14,15, 16, 17, 18, 19, 20, 21, or more) in length that is complementary toa nucleotide sequence in a target or a portion thereof (e.g., target RNAtarget). In another non-limiting example, the length of the nucleotidesequence of X and Z together (when X is present) that is complementaryto the target or a portion thereof is from about 12 to about 21 or morenucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, ormore). In yet another non-limiting example, when X is absent, the lengthof the nucleotide sequence of Z that is complementary to the target or aportion thereof is from about 12 to about 24 or more nucleotides (e.g.,about 12, 14, 16, 18, 20, 22, 24 or more). In one embodiment X, Z and X′are independently oligonucleotides, where X and/or Z comprises anucleotide sequence of length sufficient to interact (e.g., base pair)with nucleotide sequence in the target or a portion thereof (e.g.,target RNA target). In one embodiment, the lengths of oligonucleotides Xand X′ are identical. In another embodiment, the lengths ofoligonucleotides X and X′ are not identical. In another embodiment, thelengths of oligonucleotides X and Z or Z and X′ or X, Z and X′ areeither identical or different. In one embodiment, the double strandedoligonucleotide construct of Formula I(a) includes one or more,specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches donot significantly diminish the ability of the double strandedoligonucleotide to inhibit target gene expression.

In one embodiment, a DFO molecule of the invention comprises structurehaving Formula DFO-II:

5′-p-X X′-3′wherein each X and X′ are independently oligonucleotides of length about12 nucleotides to about 21 nucleotides, wherein X comprises, forexample, a nucleic acid sequence of length about 12 to about 21nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21nucleotides), X′ comprises a nucleic acid sequence, for example oflength about 12 to about 21 nucleotides (e.g., about 12, 13, 14, 15, 16,17, 18, 19, 20, or 21 nucleotides) having nucleotide sequencecomplementarity to sequence X or a portion thereof, p comprises aterminal phosphate group that can be present or absent, and wherein Xcomprises a nucleotide sequence that is complementary to a targetnucleic acid sequence (e.g., target RNA) or a portion thereof and is oflength sufficient to interact (e.g., base pair) with the target nucleicacid sequence of a portion thereof. In one embodiment, the length ofoligonucleotides X and X′ are identical. In another embodiment thelength of oligonucleotides X and X′ are not identical. In oneembodiment, length of the oligonucleotides X and X′ are sufficient toform a relatively stable double stranded oligonucleotide.

In one embodiment, the invention features a double strandedoligonucleotide construct having Formula DFO-II(a):

5′-p-X X′-3′ 3′-X′ X-p-5′wherein each X and X′ are independently oligonucleotides of length about12 nucleotides to about 21 nucleotides, wherein X comprises a nucleicacid sequence, for example of length about 12 to about 21 nucleotides(e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides), X′comprises a nucleic acid sequence, for example of length about 12 toabout 21 nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20 or21 nucleotides) having nucleotide sequence complementarity to sequence Xor a portion thereof, p comprises a terminal phosphate group that can bepresent or absent, and wherein X comprises nucleotide sequence that iscomplementary to a target nucleic acid sequence or a portion thereof(e.g., target RNA target) and is of length sufficient to interact (e.g.,base pair) with the target nucleic acid sequence (e.g., target RNA) or aportion thereof. In one embodiment, the lengths of oligonucleotides Xand X′ are identical. In another embodiment, the lengths ofoligonucleotides X and X′ are not identical. In one embodiment, thelengths of the oligonucleotides X and X′ are sufficient to form arelatively stable double stranded oligonucleotide. In one embodiment,the double stranded oligonucleotide construct of Formula II(a) includesone or more, specifically 1, 2, 3 or 4, mismatches, to the extent suchmismatches do not significantly diminish the ability of the doublestranded oligonucleotide to inhibit target gene expression.

In one embodiment, the invention features a DFO molecule having FormulaDFO-I(b):

5′-p-Z-3′.where Z comprises a palindromic or repeat nucleic acid sequenceoptionally including one or more non-standard or modified nucleotides(e.g., nucleotide with a modified base, such as 2-amino purine or auniversal base) that can facilitate base-pairing with other nucleotides.Z can be, for example, of length sufficient to interact (e.g., basepair) with nucleotide sequence of a target nucleic acid (e.g., targetRNA) molecule, preferably of length of at least 12 nucleotides,specifically about 12 to about 24 nucleotides (e.g., about 12, 14, 16,18, 20, 22 or 24 nucleotides). p represents a terminal phosphate groupthat can be present or absent.

In one embodiment, a DFO molecule having any of Formula DFO-I, DFO-I(a),DFO-I(b), DFO-II(a) or DFO-II can comprise chemical modifications asdescribed herein without limitation, such as, for example, nucleotideshaving any of Formulae I-VII, stabilization chemistries as described inTable I, or any other combination of modified nucleotides andnon-nucleotides as described in the various embodiments herein.

In one embodiment, the palindrome or repeat sequence or modifiednucleotide (e.g., nucleotide with a modified base, such as 2-aminopurine or a universal base) in Z of DFO constructs having Formula DFO-I,DFO-I(a) and DFO-I(b), comprises chemically modified nucleotides thatare able to interact with a portion of the target nucleic acid sequence(e.g., modified base analogs that can form Watson Crick base pairs ornon-Watson Crick base pairs).

In one embodiment, a DFO molecule of the invention, for example a DFOhaving Formula DFO-I or DFO-II, comprises about 15 to about 40nucleotides (e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides).In one embodiment, a DFO molecule of the invention comprises one or morechemical modifications. In a non-limiting example, the introduction ofchemically modified nucleotides and/or non-nucleotides into nucleic acidmolecules of the invention provides a powerful tool in overcomingpotential limitations of in vivo stability and bioavailability inherentto unmodified RNA molecules that are delivered exogenously. For example,the use of chemically modified nucleic acid molecules can enable a lowerdose of a particular nucleic acid molecule for a given therapeuticeffect since chemically modified nucleic acid molecules tend to have alonger half-life in serum or in cells or tissues. Furthermore, certainchemical modifications can improve the bioavailability and/or potency ofnucleic acid molecules by not only enhancing half-life but alsofacilitating the targeting of nucleic acid molecules to particularorgans, cells or tissues and/or improving cellular uptake of the nucleicacid molecules. Therefore, even if the activity of a chemically modifiednucleic acid molecule is reduced in vitro as compared to anative/unmodified nucleic acid molecule, for example when compared to anunmodified RNA molecule, the overall activity of the modified nucleicacid molecule can be greater than the native or unmodified nucleic acidmolecule due to improved stability, potency, duration of effect,bioavailability and/or delivery of the molecule.

Multifunctional or Multi-Targeted siNA Molecules of the Invention

In one embodiment, the invention features siNA molecules comprisingmultifunctional short interfering nucleic acid (multifunctional siNA)molecules that modulate the expression of one or more target genes in abiologic system, such as a cell, tissue, of organism. Themultifunctional short interfering nucleic acid (multifunctional siNA)molecules of the invention can target more than one region of the targetnucleic acid sequence or can target sequences of more than one distincttarget nucleic acid molecules (e.g., target and/or pathway target RNAand/or DNA sequences). The multifunctional siNA molecules of theinvention can be chemically synthesized or expressed from transcriptionunits and/or vectors. The multifunctional siNA molecules of the instantinvention provide useful reagents and methods for a variety of humanapplications, therapeutic, diagnostic, agricultural, veterinary, targetvalidation, genomic discovery, genetic engineering and pharmacogenomicapplications.

Applicant demonstrates herein that certain oligonucleotides, referred toherein for convenience but not limitation as multifunctional shortinterfering nucleic acid or multifunctional siNA molecules, are potentmediators of sequence specific regulation of gene expression. Themultifunctional siNA molecules of the invention are distinct from othernucleic acid sequences known in the art (e.g., siRNA, miRNA, stRNA,shRNA, antisense oligonucleotides, etc.) in that they represent a classof polynucleotide molecules that are designed such that each strand inthe multifunctional siNA construct comprises a nucleotide sequence thatis complementary to a distinct nucleic acid sequence in one or moretarget nucleic acid molecules. A single multifunctional siNA molecule(generally a double-stranded molecule) of the invention can thus targetmore than one (e.g., 2, 3, 4, 5, or more) differing target nucleic acidtarget molecules. Nucleic acid molecules of the invention can alsotarget more than one (e.g., 2, 3, 4, 5, or more) region of the sametarget nucleic acid sequence. As such multifunctional siNA molecules ofthe invention are useful in down regulating or inhibiting the expressionof one or more target nucleic acid molecules. By reducing or inhibitingexpression of more than one target nucleic acid molecule with onemultifunctional siNA construct, multifunctional siNA molecules of theinvention represent a class of potent therapeutic agents that canprovide simultaneous inhibition of multiple targets within a disease(e.g., angiogenic) related pathway. Such simultaneous inhibition canprovide synergistic therapeutic treatment strategies without the needfor separate preclinical and clinical development efforts or complexregulatory approval process.

Use of multifunctional siNA molecules that target more then one regionof a target nucleic acid molecule (e.g., target RNA or DNA) is expectedto provide potent inhibition of gene expression. For example, a singlemultifunctional siNA construct of the invention can target bothconserved and variable regions of a target nucleic acid molecule (e.g.,target RNA or DNA), thereby allowing down regulation or inhibition of,for example, different target isoforms or variants to optimizetherapeutic efficacy and minimize toxicity, or allowing for targeting ofboth coding and non-coding regions of the target nucleic acid molecule.

Generally, double stranded oligonucleotides are formed by the assemblyof two distinct oligonucleotides where the oligonucleotide sequence ofone strand is complementary to the oligonucleotide sequence of thesecond strand; such double stranded oligonucleotides are generallyassembled from two separate oligonucleotides (e.g., siRNA). Alternately,a duplex can be formed from a single molecule that folds on itself(e.g., shRNA or short hairpin RNA). These double strandedoligonucleotides are known in the art to mediate RNA interference andall have a common feature wherein only one nucleotide sequence region(guide sequence or the antisense sequence) has complementarity to atarget nucleic acid sequence, and the other strand (sense sequence)comprises nucleotide sequence that is homologous to the target nucleicacid sequence. Generally, the antisense sequence is retained in theactive RISC complex and guides the RISC to the target nucleotidesequence by means of complementary base-pairing of the antisensesequence with the target sequence for mediating sequence-specific RNAinterference. It is known in the art that in some cell culture systems,certain types of unmodified siRNAs can exhibit “off target” effects. Itis hypothesized that this off-target effect involves the participationof the sense sequence instead of the antisense sequence of the siRNA inthe RISC complex (see for example Schwarz et al., 2003, Cell, 115,199-208). In this instance the sense sequence is believed to direct theRISC complex to a sequence (off-target sequence) that is distinct fromthe intended target sequence, resulting in the inhibition of theoff-target sequence. In these double stranded nucleic acid molecules,each strand is complementary to a distinct target nucleic acid sequence.However, the off-targets that are affected by these dsRNAs are notentirely predictable and are non-specific.

Distinct from the double stranded nucleic acid molecules known in theart, the applicants have developed a novel, potentially cost effectiveand simplified method of down regulating or inhibiting the expression ofmore than one target nucleic acid sequence using a singlemultifunctional siNA construct. The multifunctional siNA molecules ofthe invention are designed to be double-stranded or partiallydouble-stranded, such that a portion of each strand or region of themultifunctional siNA is complementary to a target* nucleic acid sequenceof choice. As such, the multifunctional siNA molecules of the inventionare not limited to targeting sequences that are complementary to eachother, but rather to any two differing target nucleic acid sequences.Multifunctional siNA molecules of the invention are designed such thateach strand or region of the multifunctional siNA molecule, that iscomplementary to a given target nucleic acid sequence, is of suitablelength (e.g., from about 16 to about 28 nucleotides in length,preferably from about 18 to about 28 nucleotides in length) formediating RNA interference against the target nucleic acid sequence. Thecomplementarity between the target nucleic acid sequence and a strand orregion of the multifunctional siNA must be sufficient (at least about 8base pairs) for cleavage of the target nucleic acid sequence by RNAinterference. Multifunctional siNA of the invention is expected tominimize off-target effects seen with certain siRNA sequences, such asthose described in Schwarz et al., supra.

It has been reported that dsRNAs of length between 29 base pairs and 36base pairs (Tuschl et al., International PCT Publication No. WO02/44321) do not mediate RNAi. One reason these dsRNAs are inactive maybe the lack of turnover or dissociation of the strand that interactswith the target RNA sequence, such that the RISC complex is not able toefficiently interact with multiple copies of the target RNA resulting ina significant decrease in the potency and efficiency of the RNAiprocess. Applicant has surprisingly found that the multifunctional siNAsof the invention can overcome this hurdle and are capable of enhancingthe efficiency and potency of RNAi process. As such, in certainembodiments of the invention, multifunctional siNAs of length of about29 to about 36 base pairs can be designed such that, a portion of eachstrand of the multifunctional siNA molecule comprises a nucleotidesequence region that is complementary to a target nucleic acid of lengthsufficient to mediate RNAi efficiently (e.g., about 15 to about 23 basepairs) and a nucleotide sequence region that is not complementary to thetarget nucleic acid. By having both complementary and non-complementaryportions in each strand of the multifunctional siNA, the multifunctionalsiNA can mediate RNA interference against a target nucleic acid sequencewithout being prohibitive to turnover or dissociation (e.g., where thelength of each strand is too long to mediate RNAi against the respectivetarget nucleic acid sequence). Furthermore, design of multifunctionalsiNA molecules of the invention with internal overlapping regions allowsthe multifunctional siNA molecules to be of favorable (decreased) sizefor mediating RNA interference and of size that is well suited for useas a therapeutic agent (e.g., wherein each strand is independently fromabout 18 to about 28 nucleotides in length). Non-limiting examples areillustrated in FIGS. 16-28.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a first region and a second region, where the first region ofthe multifunctional siNA comprises a nucleotide sequence complementaryto a nucleic acid sequence of a first target nucleic acid molecule, andthe second region of the multifunctional siNA comprises nucleic acidsequence complementary to a nucleic acid sequence of a second targetnucleic acid molecule. In one embodiment, a multifunctional siNAmolecule of the invention comprises a first region and a second region,where the first region of the multifunctional siNA comprises nucleotidesequence complementary to a nucleic acid sequence of the first region ofa target nucleic acid molecule, and the second region of themultifunctional siNA comprises nucleotide sequence complementary to anucleic acid sequence of a second region of a the target nucleic acidmolecule. In another embodiment, the first region and second region ofthe multifunctional siNA can comprise separate nucleic acid sequencesthat share some degree of complementarity (e.g., from about 1 to about10 complementary nucleotides). In certain embodiments, multifunctionalsiNA constructs comprising separate nucleic acid sequences can bereadily linked post-synthetically by methods and reagents known in theart and such linked constructs are within the scope of the invention.Alternately, the first region and second region of the multifunctionalsiNA can comprise a single nucleic acid sequence having some degree ofself complementarity, such as in a hairpin or stem-loop structure.Non-limiting examples of such double stranded and hairpinmultifunctional short interfering nucleic acids are illustrated in FIGS.16 and 17 respectively. These multifunctional short interfering nucleicacids (multifunctional siNAs) can optionally include certain overlappingnucleotide sequence where such overlapping nucleotide sequence ispresent in between the first region and the second region of themultifunctional siNA (see for example FIGS. 18 and 19).

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein eachstrand of the multifunctional siNA independently comprises a firstregion of nucleic acid sequence that is complementary to a distincttarget nucleic acid sequence and the second region of nucleotidesequence that is not complementary to the target sequence. The targetnucleic acid sequence of each strand is in the same target nucleic acidmolecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence (complementaryregion 1) and a region having no sequence complementarity to the targetnucleotide sequence (non-complementary region 1); (b) the second strandof the multifunction siNA comprises a region having sequencecomplementarity to a target nucleic acid sequence that is distinct fromthe target nucleotide sequence complementary to the first strandnucleotide sequence (complementary region 2), and a region having nosequence complementarity to the target nucleotide sequence ofcomplementary region 2 (non-complementary region 2); (c) thecomplementary region 1 of the first strand comprises a nucleotidesequence that is complementary to a nucleotide sequence in thenon-complementary region 2 of the second strand and the complementaryregion 2 of the second strand comprises a nucleotide sequence that iscomplementary to a nucleotide sequence in the non-complementary region 1of the first strand. The target nucleic acid sequence of complementaryregion 1 and complementary region 2 is in the same target nucleic acidmolecule or different target nucleic acid molecules.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a gene(e.g., a first gene) (complementary region 1) and a region having nosequence complementarity to the target nucleotide sequence ofcomplementary region 1 (non-complementary region 1); (b) the secondstrand of the multifunction siNA comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a gene(e.g., a second gene) that is distinct from the gene of complementaryregion 1 (complementary region 2), and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 2 (non-complementary region 2); (c) the complementary region 1 ofthe first strand comprises a nucleotide sequence that is complementaryto a nucleotide sequence in the non-complementary region 2 of the secondstrand and the complementary region 2 of the second strand comprises anucleotide sequence that is complementary to a nucleotide sequence inthe non-complementary region 1 of the first strand.

In another embodiment, the multifunctional siNA comprises two strands,where: (a) the first strand comprises a region having sequencecomplementarity to a target nucleic acid sequence derived from a gene(e.g., gene) (complementary region 1) and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 1 (non-complementary region 1); (b) the second strand of themultifunction siNA comprises a region having sequence complementarity toa target nucleic acid sequence distinct from the target nucleic acidsequence of complementary region 1 (complementary region 2), provided,however, that the target nucleic acid sequence for complementary region1 and target nucleic acid sequence for complementary region 2 are bothderived from the same gene, and a region having no sequencecomplementarity to the target nucleotide sequence of complementaryregion 2 (non-complementary region 2); (c) the complementary region 1 ofthe first strand comprises a nucleotide sequence that is complementaryto a nucleotide sequence in the non-complementary region 2 of the secondstrand and the complementary region 2 of the second strand comprises anucleotide sequence that is complementary to nucleotide sequence in thenon-complementary region 1 of the first strand.

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having nucleotidesequence complementary to nucleotide sequence within a first targetnucleic acid molecule, and in which the second sequence comprises afirst region having nucleotide sequence complementary to a distinctnucleotide sequence within the same target nucleic acid molecule.Preferably, the first region of the first sequence is also complementaryto the nucleotide sequence of the second region of the second sequence,and where the first region of the second sequence is complementary tothe nucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional shortinterfering nucleic acid (multifunctional siNA) molecule, wherein themultifunctional siNA comprises two complementary nucleic acid sequencesin which the first sequence comprises a first region having a nucleotidesequence complementary to a nucleotide sequence within a first targetnucleic acid molecule, and in which the second sequence comprises afirst region having a nucleotide sequence complementary to a distinctnucleotide sequence within second target nucleic acid molecule.Preferably, the first region of the first sequence is also complementaryto the nucleotide sequence of the second region of the second sequence,an where the first region of the second sequence is complementary to thenucleotide sequence of the second region of the first sequence.

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises a nucleic acid sequence having about 18 to about 28nucleotides complementary to a nucleic acid sequence within a firsttarget nucleic acid molecule, and the second region comprises nucleotidesequence having about 18 to about 28 nucleotides complementary to adistinct nucleic acid sequence within a second target nucleic acidmolecule.

In one embodiment, the invention features a multifunctional siNAmolecule comprising a first region and a second region, where the firstregion comprises nucleic acid sequence having about 18 to about 28nucleotides complementary to a nucleic acid sequence within a targetnucleic acid molecule, and the second region comprises nucleotidesequence having about 18 to about 28 nucleotides complementary to adistinct nucleic acid sequence within the same target nucleic acidmolecule.

In one embodiment, the invention features a double strandedmultifunctional short interfering nucleic acid (multifunctional siNA)molecule, wherein one strand of the multifunctional siNA comprises afirst region having nucleotide sequence complementary to a first targetnucleic acid sequence, and the second strand comprises a first regionhaving a nucleotide sequence complementary to a second target nucleicacid sequence. The first and second target nucleic acid sequences can bepresent in separate target nucleic acid molecules or can be differentregions within the same target nucleic acid molecule. As such,multifunctional siNA molecules of the invention can be used to targetthe expression of different genes, splice variants of the same gene,both mutant and conserved regions of one or more gene transcripts, orboth coding and non-coding sequences of the same or differing genes orgene transcripts.

In one embodiment, a target nucleic acid molecule of the inventionencodes a single protein. In another embodiment, a target nucleic acidmolecule, encodes more than one protein (e.g., 1, 2, 3, 4, 5 or moreproteins). As such, a multifunctional siNA construct of the inventioncan be used to down regulate or inhibit the expression of severalproteins. For example, a multifunctional siNA molecule comprising aregion in one strand having nucleotide sequence complementarity to afirst target nucleic acid sequence derived from a target, and the secondstrand comprising a region with nucleotide sequence complementarity to asecond target nucleic acid sequence present in target nucleic acidmolecules from genes encoding two proteins (e.g., two differingproteins), which can be used to down regulate, inhibit, or shut down aparticular biologic pathway by targeting multiple pathway target genes.

In one embodiment the invention takes advantage of conserved nucleotidesequences present in different gene variants. By designingmultifunctional siNAs in a manner where one strand includes a sequencethat is complementary to one or more target nucleic acid sequences thatare conserved among various target gene family members and the otherstrand optionally includes sequence that is complementary to pathwaytarget nucleic acid sequence, it is possible to selectively andeffectively inhibit a target gene disease related biological pathwayusing a single multifunctional siNA.

In one embodiment, a multifunctional short interfering nucleic acid(multifunctional siNA) of the invention comprises a first region and asecond region, wherein the first region comprises nucleotide sequencecomplementary to a first target RNA of a first target and the secondregion comprises nucleotide sequence complementary to a second targetRNA of a second target. In one embodiment, the first and second regionscan comprise nucleotide sequence complementary to shared or conservedRNA sequences of differing target sites within the same target sequenceor shared amongst different target sequences.

In one embodiment, a double stranded multifunctional siNA molecule ofthe invention comprises a structure having Formula MF-I:

5′-p-X Z X′-3′ 3′-Y′ Z Y-p-5′wherein each 5′-p-XZX′-3′ and 5′-p-YZY′-3′ are independently anoligonucleotide of length about 20 nucleotides to about 300 nucleotides,preferably about 20 to about 200 nucleotides, about 20 to about 100nucleotides, about 20 to about 40 nucleotides, about 20 to about 40nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38nucleotides; XZ comprises a nucleic acid sequence that is complementaryto a first target nucleic acid sequence; YZ is an oligonucleotidecomprising nucleic acid sequence that is complementary to a secondtarget nucleic acid sequence; Z comprises nucleotide sequence of lengthabout 1 to about 24 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24nucleotides) that is self complementary; X comprises nucleotide sequenceof length about 1 to about 100 nucleotides, preferably about 1 to about21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary tonucleotide sequence present in region Y′; Y comprises nucleotidesequence of length about 1 to about 100 nucleotides, preferably about 1to about 21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that iscomplementary to nucleotide sequence present in region X; each pcomprises a terminal phosphate group that is independently present orabsent; each XZ and YZ is independently of length sufficient to stablyinteract (i.e., base pair) with the first and second target nucleic acidsequence, respectively, or a portion thereof. For example, each sequenceX and Y can independently comprise sequence from about 12 to about 21 ormore nucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19,20, 21, or more) that is complementary to a target nucleotide sequencein different target nucleic acid molecules, such as target RNAs or aportion thereof. In another non-limiting example, the length of thenucleotide sequence of X and Z together that is complementary to thefirst target nucleic acid sequence or a portion thereof is from about 12to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or more). In another non-limiting example, the length of thenucleotide sequence of Y and Z together, that is complementary to thesecond target nucleic acid sequence or a portion thereof is from about12 to about 21 or more nucleotides (e.g., about 12, 13, 14, 15, 16, 17,18, 19, 20, 21, or more). In one embodiment, the first target nucleicacid sequence and the second target nucleic acid sequence are present inthe same target nucleic acid molecule (e.g., target RNA or pathwaytarget RNA). In another embodiment, the first target nucleic acidsequence and the second target nucleic acid sequence are present indifferent target nucleic acid molecules (e.g., target RNA and pathwaytarget RNA). In one embodiment, Z comprises a palindrome or a repeatsequence. In one embodiment, the lengths of oligonucleotides X and X′are identical. In another embodiment, the lengths of oligonucleotides Xand X′ are not identical. In one embodiment, the lengths ofoligonucleotides Y and Y′ are identical. In another embodiment, thelengths of oligonucleotides Y and Y′ are not identical. In oneembodiment, the double stranded oligonucleotide construct of FormulaI(a) includes one or more, specifically 1, 2, 3 or 4, mismatches, to theextent such mismatches do not significantly diminish the ability of thedouble stranded oligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula

5′-p-X X′-3′ 3′-Y′ Y-p-5′wherein each 5′-p-XX′-3′ and 5′-p-YY′-3′ are independently anoligonucleotide of length about 20 nucleotides to about 300 nucleotides,preferably about 20 to about 200 nucleotides, about 20 to about 100nucleotides, about 20 to about 40 nucleotides, about 20 to about 40nucleotides, about 24 to about 38 nucleotides, or about 26 to about 38nucleotides; X comprises a nucleic acid sequence that is complementaryto a first target nucleic acid sequence; Y is an oligonucleotidecomprising nucleic acid sequence that is complementary to a secondtarget nucleic acid sequence; X comprises a nucleotide sequence oflength about 1 to about 100 nucleotides, preferably about 1 to about 21nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, or 21 nucleotides) that is complementary tonucleotide sequence present in region Y; Y comprises nucleotide sequenceof length about 1 to about 100 nucleotides, preferably about 1 to about21 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20 or 21 nucleotides) that is complementary tonucleotide sequence present in region X′; each p comprises a terminalphosphate group that is independently present or absent; each X and Yindependently is of length sufficient to stably interact (i.e., basepair) with the first and second target nucleic acid sequence,respectively, or a portion thereof. For example, each sequence X and Ycan independently comprise sequence from about 12 to about 21 or morenucleotides in length (e.g., about 12, 13, 14, 15, 16, 17, 18, 19, 20,21, or more) that is complementary to a target nucleotide sequence indifferent target nucleic acid molecules, such as target RNAs or aportion thereof. In one embodiment, the first target nucleic acidsequence and the second target nucleic acid sequence are present in thesame target nucleic acid molecule (e.g., target RNA or pathway targetRNA). In another embodiment, the first target nucleic acid sequence andthe second target nucleic acid sequence are present in different targetnucleic acid molecules (e.g., target RNA and pathway target RNA). In oneembodiment, Z comprises a palindrome or a repeat sequence. In oneembodiment, the lengths of oligonucleotides X and X′ are identical. Inanother embodiment, the lengths of oligonucleotides X and X′ are notidentical. In one embodiment, the lengths of oligonucleotides Y and Y′are identical. In another embodiment, the lengths of oligonucleotides Yand Y′ are not identical. In one embodiment, the double strandedoligonucleotide construct of Formula I(a) includes one or more,specifically 1, 2, 3 or 4, mismatches, to the extent such mismatches donot significantly diminish the ability of the double strandedoligonucleotide to inhibit target gene expression.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula

wherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength about 15 nucleotides to about 50 nucleotides, preferably about 18to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each X and X′is independently of length sufficient to stably interact (i.e., basepair) with a first and a second target nucleic acid sequence,respectively, or a portion thereof; W represents a nucleotide ornon-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first and second targetsequence via RNA interference. In one embodiment, the first targetnucleic acid sequence and the second target nucleic acid sequence arepresent in the same target nucleic acid molecule (e.g., target RNA orpathway target RNA). In another embodiment, the first target nucleicacid sequence and the second target nucleic acid sequence are present indifferent target nucleic acid molecules (e.g., target RNA and pathwaytarget RNA). In one embodiment, region W connects the 3′-end of sequenceY′ with the 3′-end of sequence Y. In one embodiment, region W connectsthe 3′-end of sequence Y′ with the 5′-end of sequence Y. In oneembodiment, region W connects the 5′-end of sequence Y′ with the 5′-endof sequence Y. In one embodiment, region W connects the 5′-end ofsequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminalphosphate group is present at the 5′-end of sequence X. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence X′. In one embodiment, a terminal phosphate group is present atthe 5′-end of sequence Y. In one embodiment, a terminal phosphate groupis present at the 5′-end of sequence Y′. In one embodiment, W connectssequences Y and Y′ via a biodegradable linker. In one embodiment, Wfurther comprises a conjugate, label, aptamer, ligand, lipid, orpolymer.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-IV:

wherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength about 15 nucleotides to about 50 nucleotides, preferably about 18to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each Y and Y′is independently of length sufficient to stably interact (i.e., basepair) with a first and a second target nucleic acid sequence,respectively, or a portion thereof; W represents a nucleotide ornon-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first and second targetsequence via RNA interference. In one embodiment, the first targetnucleic acid sequence and the second target nucleic acid sequence arepresent in the same target nucleic acid molecule. In another embodiment,the first target nucleic acid sequence and the second target nucleicacid sequence are present in different target nucleic acid molecules. Inone embodiment, region W connects the 3′-end of sequence Y′ with the3′-end of sequence Y. In one embodiment, region W connects the 3′-end ofsequence Y′ with the 5′-end of sequence Y. In one embodiment, region Wconnects the 5′-end of sequence Y′ with the 5′-end of sequence Y. In oneembodiment, region W connects the 5′-end of sequence Y′ with the 3′-endof sequence Y. In one embodiment, a terminal phosphate group is presentat the 5′-end of sequence X. In one embodiment, a terminal phosphategroup is present at the 5′-end of sequence X′. In one embodiment, aterminal phosphate group is present at the 5′-end of sequence Y. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence Y′. In one embodiment, W connects sequences Y and Y′ via abiodegradable linker. In one embodiment, W further comprises aconjugate, label, aptamer, ligand, lipid, or polymer.

In one embodiment, a multifunctional siNA molecule of the inventioncomprises a structure having Formula MF-V:

wherein each X, X′, Y, and Y′ is independently an oligonucleotide oflength about 15 nucleotides to about 50 nucleotides, preferably about 18to about 40 nucleotides, or about 19 to about 23 nucleotides; Xcomprises nucleotide sequence that is complementary to nucleotidesequence present in region Y′; X′ comprises nucleotide sequence that iscomplementary to nucleotide sequence present in region Y; each X, X′, Y,or Y′ is independently of length sufficient to stably interact (i.e.,base pair) with a first, second, third, or fourth target nucleic acidsequence, respectively, or a portion thereof; W represents a nucleotideor non-nucleotide linker that connects sequences Y′ and Y; and themultifunctional siNA directs cleavage of the first, second, third,and/or fourth target sequence via RNA interference. In one embodiment,the first, second, third and fourth target nucleic acid sequence are allpresent in the same target nucleic acid molecule (e.g., target RNA orpathway target RNA). In another embodiment, the first, second, third andfourth target nucleic acid sequence are independently present indifferent target nucleic acid molecules (e.g., target RNA and pathwaytarget RNA). In one embodiment, region W connects the 3′-end of sequenceY′ with the 3′-end of sequence Y. In one embodiment, region W connectsthe 3′-end of sequence Y′ with the 5′-end of sequence Y. In oneembodiment, region W connects the 5′-end of sequence Y′ with the 5′-endof sequence Y. In one embodiment, region W connects the 5′-end ofsequence Y′ with the 3′-end of sequence Y. In one embodiment, a terminalphosphate group is present at the 5′-end of sequence X. In oneembodiment, a terminal phosphate group is present at the 5′-end ofsequence X′. In one embodiment, a terminal phosphate group is present atthe 5′-end of sequence Y. In one embodiment, a terminal phosphate groupis present at the 5′-end of sequence Y′. In one embodiment, W connectssequences Y and Y′ via a biodegradable linker. In one embodiment, Wfurther comprises a conjugate, label, aptamer, ligand, lipid, orpolymer.

In one embodiment, regions X and Y of multifunctional siNA molecule ofthe invention (e.g., having any of Formula MF-I-MF-V), are complementaryto different target nucleic acid sequences that are portions of the sametarget nucleic acid molecule. In one embodiment, such target nucleicacid sequences are at different locations within the coding region of aRNA transcript. In one embodiment, such target nucleic acid sequencescomprise coding and non-coding regions of the same RNA transcript. Inone embodiment, such target nucleic acid sequences comprise regions ofalternately spliced transcripts or precursors of such alternatelyspliced transcripts.

In one embodiment, a multifunctional siNA molecule having any of FormulaMF-I-MF-V can comprise chemical modifications as described hereinwithout limitation, such as, for example, nucleotides having any ofFormulae I-VII described herein, stabilization chemistries as describedin Table I, or any other combination of modified nucleotides andnon-nucleotides as described in the various embodiments herein.

In one embodiment, the palidrome or repeat sequence or modifiednucleotide (e.g., nucleotide with a modified base, such as 2-aminopurine or a universal base) in Z of multifunctional siNA constructshaving Formula MF-I or MF-II comprises chemically modified nucleotidesthat are able to interact with a portion of the target nucleic acidsequence (e.g., modified base analogs that can form Watson Crick basepairs or non-Watson Crick base pairs).

In one embodiment, a multifunctional siNA molecule of the invention, forexample each strand of a multifunctional siNA having MF-I-MF-V,independently comprises about 15 to about 40 nucleotides (e.g., about15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, or 40 nucleotides). In one embodiment, amultifunctional siNA molecule of the invention comprises one or morechemical modifications. In a non-limiting example, the introduction ofchemically modified nucleotides and/or non-nucleotides into nucleic acidmolecules of the invention provides a powerful tool in overcomingpotential limitations of in vivo stability and bioavailability inherentto unmodified RNA molecules that are delivered exogenously. For example,the use of chemically modified nucleic acid molecules can enable a lowerdose of a particular nucleic acid molecule for a given therapeuticeffect since chemically modified nucleic acid molecules tend to have alonger half-life in serum or in cells or tissues. Furthermore, certainchemical modifications can improve the bioavailability and/or potency ofnucleic acid molecules by not only enhancing half-life but alsofacilitating the targeting of nucleic acid molecules to particularorgans, cells or tissues and/or improving cellular uptake of the nucleicacid molecules. Therefore, even if the activity of a chemically modifiednucleic acid molecule is reduced in vitro as compared to anative/unmodified nucleic acid molecule, for example when compared to anunmodified RNA molecule, the overall activity of the modified nucleicacid molecule can be greater than the native or unmodified nucleic acidmolecule due to improved stability, potency, duration of effect,bioavailability and/or delivery of the molecule.

In another embodiment, the invention features multifunctional siNAs,wherein the multifunctional siNAs are assembled from two separatedouble-stranded siNAs, with one of the ends of each sense strand istethered to the end of the sense strand of the other siNA molecule, suchthat the two antisense siNA strands are annealed to their correspondingsense strand that are tethered to each other at one end (see FIG. 22).The tethers or linkers can be nucleotide-based linkers or non-nucleotidebased linkers as generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one sense strand of the siNAis tethered to the 5′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, point away (in the opposite direction) from each other(see FIG. 22 (A)). The tethers or linkers can be nucleotide-basedlinkers or non-nucleotide based linkers as generally known in the artand as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 3′-end of one sense strand of the siNAis tethered to the 3′-end of the sense strand of the other siNAmolecule, such that the 5′-ends of the two antisense siNA strands,annealed to their corresponding sense strand that are tethered to eachother at one end, face each other (see FIG. 22 (B)). The tethers orlinkers can be nucleotide-based linkers or non-nucleotide based linkersas generally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one sense strand of the siNAis tethered to the 3′-end of the sense strand of the other siNAmolecule, such that the 5′-end of the one of the antisense siNA strandsannealed to their corresponding sense strand that are tethered to eachother at one end, faces the 3′-end of the other antisense strand (seeFIG. 22 (C-D)). The tethers or linkers can be nucleotide-based linkersor non-nucleotide based linkers as generally known in the art and asdescribed herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one antisense strand of thesiNA is tethered to the 3′-end of the antisense strand of the other siNAmolecule, such that the 5′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIG. 22 (G-H)). In one embodiment, the linkage between the 5′-endof the first antisense strand and the 3′-end of the second antisensestrand is designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterference-based cleavage of the target RNA. The tethers or linkerscan be nucleotide-based linkers or non-nucleotide based linkers asgenerally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 5′-end of one antisense strand of thesiNA is tethered to the 5′-end of the antisense strand of the other siNAmolecule, such that the 3′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIG. 22 (E)). In one embodiment, the linkage between the 5′-end ofthe first antisense strand and the 5′-end of the second antisense strandis designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′ end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterference-based cleavage of the target RNA. The tethers or linkerscan be nucleotide-based linkers or non-nucleotide based linkers asgenerally known in the art and as described herein.

In one embodiment, the invention features a multifunctional siNA,wherein the multifunctional siNA is assembled from two separatedouble-stranded siNAs, with the 3′-end of one antisense strand of thesiNA is tethered to the 3′-end of the antisense strand of the other siNAmolecule, such that the 5′-end of the one of the sense siNA strandsannealed to their corresponding antisense sense strand that are tetheredto each other at one end, faces the 3′-end of the other sense strand(see FIG. 22 (F)). In one embodiment, the linkage between the 5′-end ofthe first antisense strand and the 5′-end of the second antisense strandis designed in such a way as to be readily cleavable (e.g.,biodegradable linker) such that the 5′end of each antisense strand ofthe multifunctional siNA has a free 5′-end suitable to mediate RNAinterference-based cleavage of the target RNA. The tethers or linkerscan be nucleotide-based linkers or non-nucleotide based linkers asgenerally known in the art and as described herein.

In any of the above embodiments, a first target nucleic acid sequence orsecond target nucleic acid sequence can independently comprise targetRNA, DNA or a portion thereof. In one embodiment, the first targetnucleic acid sequence is a target RNA, DNA or a portion thereof and thesecond target nucleic acid sequence is a target RNA, DNA of a portionthereof. In one embodiment, the first target nucleic acid sequence is atarget RNA, DNA or a portion thereof and the second target nucleic acidsequence is a another RNA, DNA of a portion thereof.

Synthesis of Nucleic Acid Molecules

Synthesis of nucleic acids greater than 100 nucleotides in length isdifficult using automated methods, and the therapeutic cost of suchmolecules is prohibitive. In this invention, small nucleic acid motifs(“small” refers to nucleic acid motifs no more than 100 nucleotides inlength, preferably no more than 80 nucleotides in length, and mostpreferably no more than 50 nucleotides in length; e.g., individual siNAoligonucleotide sequences or siNA sequences synthesized in tandem) arepreferably used for exogenous delivery. The simple structure of thesemolecules increases the ability of the nucleic acid to invade targetedregions of protein and/or RNA structure. Exemplary molecules of theinstant invention are chemically synthesized, and others can similarlybe synthesized.

Oligonucleotides (e.g., certain modified oligonucleotides or portions ofoligonucleotides lacking ribonucleotides) are synthesized usingprotocols known in the art, for example as described in Caruthers etal., 1992, Methods in Enzymology 211, 3-19, Thompson et al.,International PCT Publication No. WO 99/54459, Wincott et al., 1995,Nucleic Acids Res. 23, 2677-2684, Wincott et al., 1997, Methods Mol.Bio., 74, 59, Brennan et al., 1998, Biotechnol Bioeng., 61, 33-45, andBrennan, U.S. Pat. No. 6,001,311. All of these references areincorporated herein by reference. The synthesis of oligonucleotidesmakes use of common nucleic acid protecting and coupling groups, such asdimethoxytrityl at the 5′-end, and phosphoramidites at the 3′-end. In anon-limiting example, small scale syntheses are conducted on a 394Applied Biosystems, Inc. synthesizer using a 0.2 μmol scale protocolwith a 2.5 min coupling step for 2′-O-methylated nucleotides and a 45second coupling step for 2′-deoxy nucleotides or 2′-deoxy-2′-fluoronucleotides. Table III outlines the amounts and the contact times of thereagents used in the synthesis cycle. Alternatively, syntheses at the0.2 μmol scale can be performed on a 96-well plate synthesizer, such asthe instrument produced by Protogene (Palo Alto, Calif.) with minimalmodification to the cycle. A 33-fold excess (60 μL of 0.11 M=6.6 μmol)of 2′-O-methyl phosphoramidite and a 105-fold excess of S-ethyltetrazole (60 μL of 0.25 M=15 μmol) can be used in each coupling cycleof 2′-O-methyl residues relative to polymer-bound 5′-hydroxyl. A 22-foldexcess (40 μL of 0.11 M=4.4 μmol) of deoxy phosphoramidite and a 70-foldexcess of S-ethyl tetrazole (40 μL of 0.25 M=10 μmol) can be used ineach coupling cycle of deoxy residues relative to polymer-bound5′-hydroxyl. Average coupling yields on the 394 Applied Biosystems, Inc.synthesizer, determined by colorimetric quantitation of the tritylfractions, are typically 97.5-99%. Other oligonucleotide synthesisreagents for the 394 Applied Biosystems, Inc. synthesizer include thefollowing: detritylation solution is 3% TCA in methylene chloride (ABI);capping is performed with 16% N-methyl imidazole in THF (ABI) and 10%acetic anhydride/10% 2,6-lutidine in THF (ABI); and oxidation solutionis 16.9 mM 1₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems,Inc.). Burdick & Jackson Synthesis Grade acetonitrile is used directlyfrom the reagent bottle. S-Ethyltetrazole solution (0.25 M inacetonitrile) is made up from the solid obtained from AmericanInternational Chemical, Inc. Alternately, for the introduction ofphosphorothioate linkages, Beaucage reagent (3H-1, 2-Benzodithiol-3-one1, 1-dioxide, 0.05 M in acetonitrile) is used.

Deprotection of the DNA-based oligonucleotides is performed as follows:the polymer-bound trityl-on oligoribonucleotide is transferred to a 4 mLglass screw top vial and suspended in a solution of 40% aqueousmethylamine (1 mL) at 65° C. for 10 minutes. After cooling to −20° C.,the supernatant is removed from the polymer support. The support iswashed three times with 1.0 mL of EtOH:MeCN:H2O/3:1:1, vortexed and thesupernatant is then added to the first supernatant. The combinedsupernatants, containing the oligoribonucleotide, are dried to a whitepowder. In one embodiment, the nucleic acid molecules of the inventionare synthesized, deprotected, and analyzed according to methodsdescribed in U.S. Pat. Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751,6,989,442, and U.S. Ser. No. 10/190,359, all incorporated by referenceherein in their entirety.

The method of synthesis used for RNA including certain siNA molecules ofthe invention follows the procedure as described in Usman et al., 1987,J. Am. Chem. Soc., 109, 7845; Scaringe et al., 1990, Nucleic Acids Res.,18, 5433; and Wincott et al., 1995, Nucleic Acids Res. 23, 2677-2684Wincott et al., 1997, Methods Mol. Bio., 74, 59, and makes use of commonnucleic acid protecting and coupling groups, such as dimethoxytrityl atthe 5′-end, and phosphoramidites at the 3′-end. In a non-limitingexample, small scale syntheses are conducted on a 394 AppliedBiosystems, Inc. synthesizer using a 0.2 μmol scale protocol with a 7.5min coupling step for alkylsilyl protected nucleotides and a 2.5 mincoupling step for 2′-O-methylated nucleotides. Table III outlines theamounts and the contact times of the reagents used in the synthesiscycle. Alternatively, syntheses at the 0.2 μmol scale can be done on a96-well plate synthesizer, such as the instrument produced by Protogene(Palo Alto, Calif.) with minimal modification to the cycle. A 33-foldexcess (60 μL of 0.11 M=6.6 μmol) of 2′-O-methyl phosphoramidite and a75-fold excess of S-ethyl tetrazole (60 μL of 0.25 M=15 μmol) can beused in each coupling cycle of 2′-O-methyl residues relative topolymer-bound 5′-hydroxyl. A 66-fold excess (120 μL of 0.11 M=13.2 μmol)of alkylsilyl (ribo) protected phosphoramidite and a 150-fold excess ofS-ethyl tetrazole (120 μL of 0.25 M=30 μmol) can be used in eachcoupling cycle of ribo residues relative to polymer-bound 5′-hydroxyl.Average coupling yields on the 394 Applied Biosystems, Inc. synthesizer,determined by colorimetric quantitation of the trityl fractions, aretypically 97.5-99%. Other oligonucleotide synthesis reagents for the 394Applied Biosystems, Inc. synthesizer include the following:detritylation solution is 3% TCA in methylene chloride (ABI); capping isperformed with 16% N-methyl imidazole in THF (ABI) and 10% aceticanhydride/10% 2,6-lutidine in THE (ABI); oxidation solution is 16.9 mM1₂, 49 mM pyridine, 9% water in THF (PerSeptive Biosystems, Inc.).Burdick & Jackson Synthesis Grade acetonitrile is used directly from thereagent bottle. S-Ethyltetrazole solution (0.25 M in acetonitrile) ismade up from the solid obtained from American International Chemical,Inc. Alternately, for the introduction of phosphorothioate linkages,Beaucage reagent (3H-1, 2-Benzodithiol-3-one 1, 1-dioxide 0.05 M inacetonitrile) is used.

Deprotection of the RNA is performed using either a two-pot or one-potprotocol. For the two-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 40% aq. methylamine (1 mL) at 65° C. for 10min. After cooling to −20° C., the supernatant is removed from thepolymer support. The support is washed three times with 1.0 mL ofEtOH:MeCN:H2O/3:1:1, vortexed and the supernatant is then added to thefirst supernatant. The combined supernatants, containing theoligoribonucleotide, are dried to a white powder. The base deprotectedoligoribonucleotide is resuspended in anhydrous TEA/HF/NMP solution (300μL of a solution of 1.5 mL N-methylpyrrolidinone, 750 μl TEA and 1 mLTEA.3HF to provide a 1.4 M HF concentration) and heated to 65° C. After1.5 h, the oligomer is quenched with 1.5 M NH₄HCO₃. In one embodiment,the nucleic acid molecules of the invention are synthesized,deprotected, and analyzed according to methods described in U.S. Pat.Nos. 6,995,259, 6,686,463, 6,673,918, 6,649,751, 6,989,442, and U.S.Ser. No. 10/190,359, all incorporated by reference herein in theirentirety.

Alternatively, for the one-pot protocol, the polymer-bound trityl-onoligoribonucleotide is transferred to a 4 mL glass screw top vial andsuspended in a solution of 33% ethanolic methylamine/DMSO: 1/1 (0.8 mL)at 65° C. for 15 minutes. The vial is brought to room temperatureTEA.3HF (0.1 mL) is added and the vial is heated at 65° C. for 15minutes. The sample is cooled at −20° C. and then quenched with 1.5 MNH₄HCO₃.

For purification of the trityl-on oligomers, the quenched NH₄HCO₃solution is loaded onto a C-18 containing cartridge that had beenprewashed with acetonitrile followed by 50 mM TEAA. After washing theloaded cartridge with water, the RNA is detritylated with 0.5% TFA for13 minutes. The cartridge is then washed again with water, saltexchanged with 1 M NaCl and washed with water again. The oligonucleotideis then eluted with 30% acetonitrile.

The average stepwise coupling yields are typically >98% (Wincott et al.,1995 Nucleic Acids Res. 23, 2677-2684). Those of ordinary skill in theart will recognize that the scale of synthesis can be adapted to belarger or smaller than the example described above including but notlimited to 96-well format.

Alternatively, the nucleic acid molecules of the present invention canbe synthesized separately and joined together post-synthetically, forexample, by ligation (Moore et al., 1992, Science 256, 9923; Draper etal., International PCT publication No. WO 93/23569; Shabarova et al.,1991, Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides& Nucleotides, 16, 951; Bellon et al., 1997, Bioconjugate Chem. 8, 204),or by hybridization following synthesis and/or deprotection.

The siNA molecules of the invention can also be synthesized via a tandemsynthesis methodology as described in Example 1 herein, wherein bothsiNA strands are synthesized as a single contiguous oligonucleotidefragment or strand separated by a cleavable linker which is subsequentlycleaved to provide separate siNA fragments or strands that hybridize andpermit purification of the siNA duplex. The linker can be apolynucleotide linker or a non-nucleotide linker. The tandem synthesisof siNA as described herein can be readily adapted to bothmultiwell/multiplate synthesis platforms such as 96 well or similarlylarger multi-well platforms. The tandem synthesis of siNA as describedherein can also be readily adapted to large scale synthesis platformsemploying batch reactors, synthesis columns and the like.

A siNA molecule can also be assembled from two distinct nucleic acidstrands or fragments wherein one fragment includes the sense region andthe second fragment includes the antisense region of the RNA molecule.

The nucleic acid molecules of the present invention can be modifiedextensively to enhance stability by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H(for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al.,1994, Nucleic Acids Symp. Ser. 31, 163). siNA constructs can be purifiedby gel electrophoresis using general methods or can be purified by highpressure liquid chromatography (HPLC; see Wincott et al., supra, thetotality, of which is hereby incorporated herein by reference) andre-suspended in water.

In another aspect of the invention, siNA molecules of the invention areexpressed from transcription units inserted into DNA or RNA vectors. Therecombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Therecombinant vectors capable of expressing the siNA molecules can bedelivered as described herein, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of siNA molecules.

Optimizing Activity of the Nucleic Acid Molecule of the Invention.

Chemically synthesizing nucleic acid molecules with modifications (base,sugar and/or phosphate) can prevent their degradation by serumribonucleases, which can increase their potency (see e.g., Eckstein etal., International Publication No. WO 92/07065; Perrault et al., 1990Nature 344, 565; Pieken et al., 1991, Science 253, 314; Usman andCedergren, 1992, Trends in Biochem. Sci. 17, 334; Usman et al.,International Publication No. WO 93/15187; and Rossi et al.,International Publication No. WO 91/03162; Sproat, U.S. Pat. No.5,334,711; Gold et al., U.S. Pat. No. 6,300,074; and Burgin et al.,supra; all of which are incorporated by reference herein). All of theabove references describe various chemical modifications that can bemade to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

There are several examples in the art describing sugar, base andphosphate modifications that can be introduced into nucleic acidmolecules with significant enhancement in their nuclease stability andefficacy. For example, oligonucleotides are modified to enhancestability and/or enhance biological activity by modification withnuclease resistant groups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro,2′-O-methyl, 2′-O-allyl, 2′-H, nucleotide base modifications (for areview see Usman and Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994,Nucleic Acids Symp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35,14090). Sugar modification of nucleic acid molecules have beenextensively described in the art (see Eckstein et al., InternationalPublication PCT No. WO 92/07065; Perrault et al. Nature, 1990, 344,565-568; Pieken et al. Science, 1991, 253, 314-317; Usman and Cedergren,Trends in Biochem. Sci., 1992, 17, 334-339; Usman et al. InternationalPublication PCT No. WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 andBeigelman et al., 1995, J. Biol. Chem., 270, 25702; Beigelman et al.,International PCT publication No. WO 97/26270; Beigelman et al., U.S.Pat. No. 5,716,824; Usman et al., U.S. Pat. No. 5,627,053; Woolf et al.,International PCT Publication No. WO 98/13526; Thompson et al., U.S.Ser. No. 60/082,404 which was filed on Apr. 20, 1998; Karpeisky et al.,1998, Tetrahedron Lett., 39, 1131; Earnshaw and Gait, 1998, Biopolymers(Nucleic Acid Sciences), 48, 39-55; Verma and Eckstein, 1998, Annu. Rev.Biochem., 67, 99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5,1999-2010; all of the references are hereby incorporated in theirtotality by reference herein). Such publications describe generalmethods and strategies to determine the location of incorporation ofsugar, base and/or phosphate modifications and the like into nucleicacid molecules without modulating catalysis, and are incorporated byreference herein. In view of such teachings, similar modifications canbe used as described herein to modify the siNA nucleic acid molecules ofthe instant invention so long as the ability of siNA to promote RNAi iscells is not significantly inhibited.

In one embodiment, a nucleic acid molecule of the invention ischemically modified as described in US 20050020521, incorporated byreference herein in its entirety.

While chemical modification of oligonucleotide internucleotide linkageswith phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonatelinkages improves stability, excessive modifications can cause sometoxicity or decreased activity. Therefore, when designing nucleic acidmolecules, the amount of these internucleotide linkages should beminimized. The reduction in the concentration of these linkages shouldlower toxicity, resulting in increased efficacy and higher specificityof these molecules.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are provided. Such anucleic acid is also generally more resistant to nucleases than anunmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. In cases in whichmodulation is the goal, therapeutic nucleic acid molecules deliveredexogenously should optimally be stable within cells until translation ofthe target RNA has been modulated long enough to reduce the levels ofthe undesirable protein. This period of time varies between hours todays depending upon the disease state. Improvements in the chemicalsynthesis of RNA and DNA (Wincott et al., 1995, Nucleic Acids Res. 23,2677; Caruthers et al., 1992, Methods in Enzymology 211, 3-19(incorporated by reference herein)) have expanded the ability to modifynucleic acid molecules by introducing nucleotide modifications toenhance their nuclease stability, as described above.

In one embodiment, nucleic acid molecules of the invention include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clampnucleotides. A G-clamp nucleotide is a modified cytosine analog whereinthe modifications confer the ability to hydrogen bond both Watson-Crickand Hoogsteen faces of a complementary guanine within a duplex, see forexample Lin and Matteucci, 1998, J. Am. Chem. Soc., 120, 8531-8532. Asingle G-clamp analog substitution within an oligonucleotide can resultin substantially enhanced helical thermal stability and mismatchdiscrimination when hybridized to complementary oligonucleotides. Theinclusion of such nucleotides in nucleic acid molecules of the inventionresults in both enhanced affinity and specificity to nucleic acidtargets, complementary sequences, or template strands. In anotherembodiment, nucleic acid molecules of the invention include one or more(e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “locked nucleicacid” nucleotides such as a 2′, 4′-C methylene bicyclo nucleotide (seefor example Wengel et al., International PCT Publication No. WO 00/66604and WO 99/14226).

In another embodiment, the invention features conjugates and/orcomplexes of siNA molecules of the invention. Such conjugates and/orcomplexes can be used to facilitate delivery of siNA molecules into abiological system, such as a cell. The conjugates and complexes providedby the instant invention can impart therapeutic activity by transferringtherapeutic compounds across cellular membranes, altering thepharmacokinetics, and/or modulating the localization of nucleic acidmolecules of the invention. The present invention encompasses the designand synthesis of novel conjugates and complexes for the delivery ofmolecules, including, but not limited to, small molecules, lipids,cholesterol, phospholipids, nucleosides, nucleotides, nucleic acids,antibodies, toxins, negatively charged polymers and other polymers, forexample proteins, peptides, hormones, carbohydrates, polyethyleneglycols, or polyamines, across cellular membranes. In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds are expected to improve delivery and/or localization ofnucleic acid molecules of the invention into a number of cell typesoriginating from different tissues, in the presence or absence of serum(see Sullenger and Cech, U.S. Pat. No. 5,854,038). Conjugates of themolecules described herein can be attached to biologically activemolecules via linkers that are biodegradable, such as biodegradablenucleic acid linker molecules.

The term “biodegradable linker” as used herein, refers to a nucleic acidor non nucleic acid linker molecule that is designed as a biodegradablelinker to connect one molecule to another molecule, for example, abiologically active molecule to a siNA molecule of the invention or thesense and antisense stranls of a siNA molecule of the invention. Thebiodegradable linker is designed such that its stability can bemodulated for a particular purpose, such as delivery to a particulartissue or cell type. The stability of a nucleic acid-based biodegradablelinker molecule can be modulated by using various chemistries, forexample combinations of ribonucleotides, deoxyribonucleotides, andchemically-modified nucleotides, such as 2′-O-methyl, 2′-fluoro,2′-amino, 2′-O-amino, 2′-C-allyl, 2′-O-allyl, and other 2′-modified orbase modified nucleotides. The biodegradable nucleic acid linkermolecule can be a dimer, trimer, tetramer or longer nucleic acidmolecule, for example, an oligonucleotide of about 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length,or can comprise a single nucleotide with a phosphorus-based linkage, forexample, a phosphoramidate or phosphodiester linkage. The biodegradablenucleic acid linker molecule can also comprise nucleic acid backbone,nucleic acid sugar, or nucleic acid base modifications.

The term “biodegradable” as used herein, refers to degradation in abiological system, for example, enzymatic degradation or chemicaldegradation.

The term “biologically active molecule” as used herein refers tocompounds or molecules that are capable of eliciting or modifying abiological response in a system. Non-limiting examples of biologicallyactive siNA molecules either alone or in combination with othermolecules contemplated by the instant invention include therapeuticallyactive molecules such as antibodies, cholesterol, hormones, antivirals,peptides, proteins, chemotherapeutics, small molecules, vitamins,co-factors, nucleosides, nucleotides, oligonucleotides, enzymaticnucleic acids, antisense nucleic acids, triplex formingoligonucleotides, 2,5-A chimeras, siNA, dsRNA, allozymes, aptamers,decoys and analogs thereof. Biologically active molecules of theinvention also include molecules capable of modulating thepharmacokinetics and/or pharmacodynamics of other biologically activemolecules, for example, lipids and polymers such as polyamines,polyamides, polyethylene glycol and other polyethers.

The term “phospholipid” as used herein, refers to a hydrophobic moleculecomprising at least one phosphorus group. For example, a phospholipidcan comprise a phosphorus-containing group and saturated or unsaturatedalkyl group, optionally substituted with OH, COOH, oxo, amine, orsubstituted or unsubstituted aryl groups.

Therapeutic nucleic acid molecules (e.g., siNA molecules) deliveredexogenously optimally are stable within cells until reversetranscription of the RNA has been modulated long enough to reduce thelevels of the RNA transcript. The nucleic acid molecules are resistantto nucleases in order to function as effective intracellular therapeuticagents. Improvements in the chemical synthesis of nucleic acid moleculesdescribed in the instant invention and in the art have expanded theability to modify nucleic acid molecules by introducing nucleotidemodifications to enhance their nuclease stability as described above.

In yet another embodiment, siNA molecules having chemical modificationsthat maintain or enhance enzymatic activity of proteins involved in RNAiare provided. Such nucleic acids are also generally more resistant tonucleases than unmodified nucleic acids. Thus, in vitro and/or in vivothe activity should not be significantly lowered.

Use of the nucleic acid-based molecules of the invention will lead tobetter treatments by affording the possibility of combination therapies(e.g., multiple siNA molecules targeted to different genes; nucleic acidmolecules coupled with known small molecule modulators; or intermittenttreatment with combinations of molecules, including different motifsand/or other chemical or biological molecules). The treatment ofsubjects with siNA molecules can also include combinations of differenttypes of nucleic acid molecules, such as enzymatic nucleic acidmolecules (ribozymes), allozymes, antisense, 2,5-A oligoadenylate,decoys, and aptamers.

In another aspect a siNA molecule of the invention comprises one or more5′ and/or a 3′-cap structure, for ‘example, on only the sense siNAstrand, the antisense siNA strand, or both siNA strands.

By “cap structure” is meant chemical modifications, which have beenincorporated at either terminus of the oligonucleotide (see, forexample, Adamic et al., U.S. Pat. No. 5,998,203, incorporated byreference herein). These terminal modifications protect the nucleic acidmolecule from exonuclease degradation, and may help in delivery and/orlocalization within a cell. The cap may be present at the 5′-terminus(5′-cap) or at the 3′-terminal (3′-cap) or may be present on bothtermini. In non-limiting examples, the 5′-cap includes, but is notlimited to, glyceryl, inverted deoxy abasic residue (moiety);4′,5′-methylene nucleotide; 1-(beta-D-erythrofuranosyl) nucleotide,4′-thio nucleotide; carbocyclic nucleotide; 1,5-anhydrohexitolnucleotide; L-nucleotides; alpha-nucleotides; modified base nucleotide;phosphorodithioate linkage; threo-pentofuranosyl nucleotide; acyclic3′,4′-seco nucleotide; acyclic 3,4-dihydroxybutyl nucleotide; acyclic3,5-dihydroxypentyl nucleotide, 3′-3′-inverted nucleotide moiety;3′-3′-inverted abasic moiety; 3′-2′-inverted nucleotide moiety;3′-2′-inverted abasic moiety; 1,4-butanediol phosphate;3′-phosphoramidate; hexylphosphate; aminohexyl phosphate; 3′-phosphate;3′-phosphorothioate; phosphorodithioate; or bridging or non-bridgingmethylphosphonate moiety. Non-limiting examples of cap moieties areshown in FIG. 10.

Non-limiting examples of the 3′-cap include, but are not limited to,glyceryl, inverted deoxy abasic residue (moiety), 4′, 5′-methylenenucleotide; 1-(b eta-D-erythrofuranosyl) nucleotide; 4′-thio nucleotide,carbocyclic nucleotide; 5′-amino-alkyl phosphate; 1,3-diamino-2-propylphosphate; 3-aminopropyl phosphate; 6-aminohexyl phosphate;1,2-arninododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitolnucleotide; L-nucleotide; alpha-nucleotide; modified base nucleotide;phosphorodithioate; threo-pentofuranosyl nucleotide; acyclic 3′,4′-seconucleotide; 3,4-dihydroxybutyl nucleotide; 3,5-dihydroxypentylnucleotide, 5′-5′-inverted nucleotide moiety; 5′-5′-inverted abasicmoiety; 5′-phosphoramidate; 5′-phosphorothioate; 1,4-butanediolphosphate; 5′-amino; bridging and/or non-bridging 5′-phosphoramidate,phosphorothioate and/or phosphorodithioate, bridging or non bridgingmethylphosphonate and 5′-mercapto moieties (for more details seeBeaucage and Iyer, 1993, Tetrahedron 49, 1925; incorporated by referenceherein).

By the term “non-nucleotide” is meant any group or compound which can beincorporated into a nucleic acid chain in the place of one or morenucleotide units, including either sugar and/or phosphate substitutions,and allows the remaining bases to exhibit their enzymatic activity. Thegroup or compound is abasic in that it does not contain a commonlyrecognized nucleotide base, such as adenosine, guanine, cytosine, uracilor thymine and therefore lacks a base at the 1′-position.

An “alkyl” group refers to a saturated aliphatic hydrocarbon, includingstraight chain, branched-chain, and cyclic alkyl groups. Preferably, thealkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl offrom 1 to 7 carbons, more preferably 1 to 4 carbons. The alkyl group canbe substituted or unsubstituted. When substituted the substitutedgroup(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂,amino, or SH. The term also includes alkenyl groups that are unsaturatedhydrocarbon groups containing at least one carbon-carbon double bond,including straight-chain, branched-chain, and cyclic groups. Preferably,the alkenyl group has 1 to 12 carbons. More preferably, it is a loweralkenyl of from 1 to 7 carbons, more preferably 1 to 4 carbons. Thealkenyl group may be substituted or unsubstituted. When substituted thesubstituted group(s) is preferably, hydroxyl, cyano, alkoxy, ═O, ═S,NO₂, halogen, N(CH₃)₂, amino, or SH. The term “alkyl” also includesalkynyl groups that have an unsaturated hydrocarbon group containing atleast one carbon-carbon triple bond, including straight-chain,branched-chain, and cyclic groups. Preferably, the alkynyl group has 1to 12 carbons. More preferably, it is a lower alkynyl of from 1 to 7carbons, more preferably 1 to 4 carbons. The alkynyl group may besubstituted or unsubstituted. When substituted the substituted group(s)is preferably, hydroxyl, cyano, alkoxy, ═O, ═S, NO₂ or N(CH₃)₂, amino orSH.

Such alkyl groups can also include aryl, alkylaryl, carbocyclic aryl,heterocyclic aryl, amide and ester groups. An “aryl” group refers to anaromatic group that has at least one ring having a conjugated pielectron system and includes carbocyclic aryl, heterocyclic aryl andbiaryl groups, all of which may be optionally substituted. The preferredsubstituent(s) of aryl groups are halogen, trihalomethyl, hydroxyl, SH,OH, cyan, alkoxy, alkyl, alkenyl, alkynyl, and amino groups. An“alkylaryl” group refers to an alkyl group (as described above)covalently joined to an aryl group (as described above). Carbocyclicaryl groups are groups wherein the ring atoms on the aromatic ring areall carbon atoms. The carbon atoms are optionally substituted.Heterocyclic aryl groups are groups having from 1 to 3 heteroatoms asring atoms in the aromatic ring and the remainder of the ring atoms arecarbon atoms. Suitable heteroatoms include oxygen, sulfur, and nitrogen,and include furanyl, thienyl, pyridyl, pyrrolyl, N-lower alkyl pyrrolo,pyrimidyl, pyrazinyl, imidazolyl and the like, all optionallysubstituted. An “amide” refers to an —C(O)—NH—R, where R is eitheralkyl, aryl, alkylaryl or hydrogen. An “ester” refers to an —C(O)—OR′,where R is either alkyl, aryl, alkylaryl or hydrogen.

By “nucleotide” as used herein is as recognized in the art to includenatural bases (standard), and modified bases well known in the art. Suchbases are generally located at the 1 position of a nucleotide sugarmoiety. Nucleotides generally comprise a base, sugar and a phosphategroup. The nucleotides can be unmodified or modified at the sugar,phosphate and/or base moiety, (also referred to interchangeably asnucleotide analogs, modified nucleotides, non-natural nucleotides,non-standard nucleotides and other; see, for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of base modifications that canbe introduced into nucleic acid molecules include, inosine, purine,pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2, 4, 6-trimethoxybenzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl,5-alkylcytidines (e.g., 5-methylcytidine), 5-alkyluridines (e.g.,ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidinesor 6-alkylpyrimidines (e.g., 6-methyluridine), propyne, and others(Burgin et al., 1996, Biochemistry, 35, 14090; Uhlman & Peyman, supra).By “modified bases” in this aspect is meant nucleotide bases other thanadenine, guanine, cytosine and uracil at 1′ position or theirequivalents.

In one embodiment, the invention features modified siNA molecules, withphosphate backbone modifications comprising one or morephosphorothioate, phosphorodithioate, methylphosphonate,phosphotriester, morpholino, amidate carbamate, carboxymethyl,acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal,thioformacetal, and/or alkylsilyl, substitutions. For a review ofoligonucleotide backbone modifications, see Hunziker and Leumann, 1995,Nucleic Acid Analogues: Synthesis and Properties, in Modern SyntheticMethods, VCH, 331-417, and Mesmaeker et al., 1994, Novel BackboneReplacements for Oligonucleotides, in Carbohydrate Modifications inAntisense Research, ACS, 24-39.

By “abasic” is meant sugar moieties lacking a nucleobase or having ahydrogen atom (H) or other non-nucleobase chemical groups in place of anucleobase at the 1′ position of the sugar moiety, see for exampleAdamic et al., U.S. Pat. No. 5,998,203. In one embodiment, an abasicmoiety of the invention is a ribose, deoxyribose, or dideoxyribosesugar.

By “unmodified nucleoside” is meant one of the bases adenine, cytosine,guanine, thymine, or uracil joined to the 1′ carbon ofβ-D-ribo-furanose.

By “modified nucleoside” is meant any nucleotide base which contains amodification in the chemical structure of an unmodified nucleotide base,sugar and/or phosphate. Non-limiting examples of modified nucleotidesare shown by Formulae 1-VII and/or other modifications described herein.

In connection with 2′-modified nucleotides as described for the presentinvention, by “amino” is meant 2′-NH₂ or 2′-O—NH₂, which can be modifiedor unmodified. Such modified groups are described, for example, inEckstein et al., U.S. Pat. No. 5,672,695 and Matulic-Adamic et al., U.S.Pat. No. 6,248,878, which are both incorporated by reference in theirentireties.

Various modifications to nucleic acid siNA structure can be made toenhance the utility of these molecules. Such modifications will enhanceshelf-life, half-life in vitro, stability, and ease of introduction ofsuch oligonucleotides to the target site, e.g., to enhance penetrationof cellular membranes, and confer the ability to recognize and bind totargeted cells.

Administration of Nucleic Acid Molecules

A siNA molecule of the invention can be adapted for use to prevent ortreat diseases, traits, disorders, and/or conditions described herein orotherwise known in the art to be related to target gene or targetpathway gene expression, and/or any other trait, disease, disorder orcondition that is related to or will respond to the levels of targetpolynucleotides or proteins expressed therefrom in a cell or tissue,alone or in combination with other therapies. In one embodiment, thesiNA molecules of the invention and formulations or compositions thereofare administered to a cell, subject, or organism as is described hereinand as is generally known in the art.

In one embodiment, a siNA composition of the invention can comprise adelivery vehicle, including liposomes, for administration to a subject,carriers and diluents and their salts, and/or can be present inpharmaceutically acceptable formulations. Methods for the delivery ofnucleic acid molecules are described in Akhtar et al., 1992, Trends CellBio., 2, 139; Delivery Strategies for Antisense OligonucleotideTherapeutics, ed. Akhtar, 1995, Maurer et al., 1999, Mol. Membr. Biol.,16, 129-140; Hofland and Huang, 1999, Handb. Exp. Pharmacol., 137,165-192; and Lee et al., 2000, ACS Symp. Ser., 752, 184-192, all ofwhich are incorporated herein by reference. Beigelman et al., U.S. Pat.No. 6,395,713 and Sullivan et al., PCT WO 94/02595 further describe thegeneral methods for delivery of nucleic acid molecules. These protocolscan be utilized for the delivery of virtually any nucleic acid molecule.Nucleic acid molecules can be administered to cells by a variety ofmethods known to those of skill in the art, including, but notrestricted to, encapsulation in liposomes, by iontophoresis, or byincorporation into other vehicles, such as biodegradable polymers,hydrogels, cyclodextrins (see for example Gonzalez et al., 1999,Bioconjugate Chem., 10, 1068-1074; Wang et al., International PCTpublication Nos. WO 03/47518 and WO 03/46185),poly(lactic-co-glycolic)acid (PLGA) and PLCA microspheres (see forexample U.S. Pat. No. 6,447,796 and US Patent Application PublicationNo. US 2002130430), biodegradable nanocapsules, and bioadhesivemicrospheres, or by proteinaceous vectors (O'Hare and Normand,International PCT Publication No. WO 00/53722). In another embodiment,the nucleic acid molecules of the invention can also be formulated orcomplexed with polyethyleneimine and derivatives thereof, such aspolyethyleneimine-polyethyleneglycol-N-acetylgalactosamine (PEI-PEG-GAL)or polyethyleneimine polyethyleneglycol-tri-N-acetylgalactosamine(PEI-PEG-triGAL) derivatives. In one embodiment, the nucleic acidmolecules of the invention are formulated as described in United StatesPatent Application Publication No. 20030077829, incorporated byreference herein in its entirety.

In one embodiment, a siNA molecule of the invention is formulated as acomposition described in U.S. Provisional patent application No.60/678,531 and in related U.S. Provisional patent application No.60/703,946, filed Jul. 29, 2005, U.S. Provisional patent application No.60/737,024, filed Nov. 15, 2005, and U.S. Ser. No. 11/353,630, filedFeb. 14, 2006 (Vargeese et al.), all of which are incorporated byreference herein in their entirety. Such siNA formulations are generallyreferred to as “lipid nucleic acid particles” (LNP). In one embodiment,a siNA molecule of the invention is formulated with one or more LNPcompositions described herein in Table IV (see U.S. Ser. No. 11/353,630supra).

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to lung tissues and cells as isdescribed in US 2006/0062758; US 2006/0014289; and US 2004/0077540.

In one embodiment, a siNA molecule of the invention is complexed withmembrane disruptive agents such as those described in U.S. PatentApplication Publication No. 20010007666, incorporated by referenceherein in its entirety including the drawings. In another embodiment,the membrane disruptive agent or agents and the siNA molecule are alsocomplexed with a cationic lipid or helper lipid molecule, such as thoselipids described in U.S. Pat. No. 6,235,310, incorporated by referenceherein in its entirety including the drawings.

In one embodiment, a siNA molecule of the invention is complexed withdelivery systems as described in U.S. Patent Application Publication No.2003077829 and International PCT Publication Nos. WO 00/03683 and WO02/087541, all incorporated by reference herein in their entiretyincluding the drawings.

In one embodiment, a siNA molecule of the invention is complexed withdelivery systems as is generally described in U.S. Patent ApplicationPublication Nos. US-20050287551; US-20050164220; US-20050191627;US-20050118594; US-20050153919; US-20050085486; and US-20030158133; allincorporated by reference herein in their entirety including thedrawings.

In one embodiment, the nucleic acid molecules of the invention areadministered to skeletal tissues (e.g., bone, cartilage, tendon,ligament) or bone metastatic tumors via atelocollagen complexation orconjugation (see for example Takeshita et al., 2005, PNAS, 102,12177-12182). Therefore, in one embodiment, the instant inventionfeatures one or more dsiNA molecules as a composition complexed withatelocollagen. In another embodiment, the instant invention features oneor more siNA molecules conjugated to atelocollagen via a linker asdescribed herein or otherwise known in the art.

In one embodiment, the nucleic acid molecules of the invention andformulations thereof (e.g., LNP formulations of double stranded nucleicacid molecules of the invention) are administered via pulmonarydelivery, such as by inhalation of an aerosol or spray dried formulationadministered by an inhalation device or nebulizer, providing rapid localuptake of the nucleic acid molecules into relevant pulmonary tissues.Solid particulate compositions containing respirable dry particles ofmicronized nucleic acid compositions can be prepared by grinding driedor lyophilized nucleic acid compositions, and then passing themicronized composition through, for example, a 400 mesh screen to breakup or separate out large agglomerates. A solid particulate compositioncomprising the nucleic acid compositions of the invention can optionallycontain a dispersant which serves to facilitate the formation of anaerosol as well as other therapeutic compounds. A suitable dispersant islactose, which can be blended with the nucleic acid compound in anysuitable ratio, such as a 1 to 1 ratio by weight.

Aerosols of liquid particles comprising a nucleic acid composition ofthe invention can be produced by any suitable means, such as with anebulizer (see for example U.S. Pat. No. 4,501,729). Nebulizers arecommercially available devices which transform solutions or suspensionsof an active ingredient into a therapeutic aerosol mist either by meansof acceleration of a compressed gas, typically air or oxygen, through anarrow venturi orifice or by means of ultrasonic agitation. Suitableformulations for use in nebulizers comprise the active ingredient in aliquid carrier in an amount of up to 40% w/w preferably less than 20%w/w of the formulation. The carrier is typically water or a diluteaqueous alcoholic solution, preferably made isotonic with body fluids bythe addition of, for example, sodium chloride or other suitable salts.Optional additives include preservatives if the formulation is notprepared sterile, for example, methyl hydroxybenzoate, anti-oxidants,flavorings, volatile oils, buffering agents and emulsifiers and otherformulation surfactants. The aerosols of solid particles comprising theactive composition and surfactant can likewise be produced with anysolid particulate aerosol generator. Aerosol generators foradministering solid particulate therapeutics to a subject produceparticles which are respirable, as explained above, and generate avolume of aerosol containing a predetermined metered dose of atherapeutic composition at a rate suitable for human administration.

In one embodiment, a solid particulate aerosol generator of theinvention is an insufflator. Suitable formulations for administration byinsufflation include finely comminuted powders which can be delivered bymeans of an insufflator. In the insufflator, the powder, e.g., a metereddose thereof effective to carry out the treatments described herein, iscontained in capsules or cartridges, typically made of gelatin orplastic, which are either pierced or opened in situ and the powderdelivered by air drawn through the device upon inhalation or by means ofa manually-operated pump. The powder employed in the insufflatorconsists either solely of the active ingredient or of a powder blendcomprising the active ingredient, a suitable powder diluent, such aslactose, and an optional surfactant. The active ingredient typicallycomprises from 0.1 to 100 w/w of the formulation. A second type ofillustrative aerosol generator comprises a metered dose inhaler. Metereddose inhalers are pressurized aerosol dispensers, typically containing asuspension or solution formulation of the active ingredient in aliquified propellant. During use these devices discharge the formulationthrough a valve adapted to deliver a metered volume to produce a fineparticle spray containing the active ingredient. Suitable propellantsinclude certain chlorofluorocarbon compounds, for example,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane and mixtures thereof. The formulation canadditionally contain one or more co-solvents, for example, ethanol,emulsifiers and other formulation surfactants, such as oleic acid orsorbitan trioleate, anti-oxidants and suitable flavoring agents. Othermethods for pulmonary delivery are described in, for example US PatentApplication No. 20040037780, and U.S. Pat. Nos. 6,592,904; 6,582,728;6,565,885, all incorporated by reference herein.

In one embodiment, the siNA and LNP compositions and formulationsprovided herein for use in pulmonary delivery further comprise one ormore surfactants. Suitable surfactants or surfactant components forenhancing the uptake of the compositions of the invention includesynthetic and natural as well as full and truncated forms of surfactantprotein A, surfactant protein B, surfactant protein C, surfactantprotein D and surfactant Protein E, di-saturated phosphatidylcholine(other than dipalmitoyl), dipalmitoylphosphatidylchol-ine,phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol,phosphatidylethanolamine, phosphatidylserine; phosphatidic acid,ubiquinones, lysophosphatidylethanolamine, lysophosphatidylcholine,palmitoyl-lysophosphatidylcholine, dehydroepiandrosterone, dolichols,sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate,glycerol, glycero-3-pho phocholine, dihydroxyacetone, palmitate,cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline, cholinephosphate; as well as natural and artificial lamelar bodies which arethe natural carrier vehicles for the components of surfactant, omega-3fatty acids, polyenic acid, polyenoic acid, lecithin, palmitinic acid,non-ionic block copolymers of ethylene or propylene oxides,polyoxypropylene, monomeric and polymeric, polyoxyethylene, monomericand polymeric, poly (vinyl amine) with dextran and/or alkanoyl sidechains, Brij 35, Triton X-100 and synthetic surfactants ALEC, Exosurf,Survan and Atovaquone, among others. These surfactants may be useedeither as single or part of a multiple component surfactant in aformulation, or as covalently bound additions to the 5′ and/or 3′ endsof the nucleic acid component of a pharmaceutical composition herein.

The composition of the present invention may be administered into therespiratory system as a formulation including particles of respirablesize, e.g., particles of a size sufficiently small to pass through thenose, mouth and larynx upon inhalation and through the bronchi andalveoli of the lungs. In general, respirable particles range from about0.5 to 10 microns in size. Particles of non-respirable size which areincluded in the aerosol tend to deposit in the throat and be swallowed,and the quantity of non-respirable particles in the aerosol is thusminimized. For nasal administration, a particle size in the range of10-500 um is preferred to ensure retention in the nasal cavity.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered to the liver as is generallyknown in the art (see for example Wen et al., 2004, World JGastroenterol., 10, 244-9; Murao et al., 2002, Pharm Res., 19, 1808-14;Liu et al., 2003, gene Ther., 10, 180-7; Hong et al., 2003, J PharmPharmacol., 54, 51-8; Hellmann et al., 2004, Arch Virol., 149, 1611-7;and Matsuno et al., 2003, gene Ther., 10, 1559-66).

In one embodiment, the invention features the use of methods to deliverthe nucleic acid molecules of the instant invention to the centralnervous system and/or peripheral nervous system. Experiments havedemonstrated the efficient in vivo uptake of nucleic acids by neurons.As an example of local administration of nucleic acids to nerve cells,Sommer et al., 1998, Antisense Nuc. Acid Drug Dev., 8, 75, describe astudy in which a 15mer phosphorothioate antisense nucleic acid moleculeto c-fos is administered to rats via microinjection into the brain.Antisense molecules labeled with tetramethylrhodamine-isothiocyanate(TRITC) or fluorescein isothiocyanate (FITC) were taken up byexclusively by neurons thirty minutes post-injection. A diffusecytoplasmic staining and nuclear staining was observed in these cells.As an example of systemic administration of nucleic acid to nerve cells,Epa et al., 2000, Antisense Nuc. Acid Drug Dev., 10, 469, describe an invivo mouse study in which beta-cyclodextrin-adamantane-oligonucleotideconjugates were used to target the p75 neurotrophin receptor inneuronally differentiated PC12 cells. Following a two week course of IPadministration, pronounced uptake of p75 neurotrophin receptor antisensewas observed in dorsal root ganglion (DRG) cells. In addition, a markedand consistent down-regulation of p75 was observed in DRG neurons.Additional approaches to the targeting of nucleic acid to neurons aredescribed in Broaddus et al., 1998, J. Neurosurg., 88(4), 734; Karle etal., 1997, Eur. J. Pharinocol., 340(2/3), 153; Bannai et al., 1998,Brain Research, 784(1,2), 304; Raj akumar et al., 1997, Synapse, 26(3),199; Wu-pong et al., 1999, BioPharm, 12(1), 32; Bannai et al., 1998,Brain Res. Protoc., 3(1), 83; Simantov et al., 1996, Neuroscience,74(1), 39. Nucleic acid molecules of the invention are thereforeamenable to delivery to and uptake by cells that express repeatexpansion allelic variants for modulation of RE gene expression. Thedelivery of nucleic acid molecules of the invention, targeting RE isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

The delivery of nucleic acid molecules of the invention to the CNS isprovided by a variety of different strategies. Traditional approaches toCNS delivery that can be used include, but are not limited to,intrathecal and intracerebroventricular administration, implantation ofcatheters and pumps, direct injection or perfusion at the site of injuryor lesion, injection into the brain arterial system, or by chemical orosmotic opening of the blood-brain barrier. Other approaches can includethe use of various transport and carrier systems, for example though theuse of conjugates and biodegradable polymers. Furthermore, gene therapyapproaches, for example as described in Kaplitt et al., U.S. Pat. No.6,180,613 and Davidson, WO 04/013280, can be used to express nucleicacid molecules in the CNS.

In one embodiment, a compound, molecule, or composition for thetreatment of ocular conditions (e.g., macular degeneration, diabeticretinopathy etc.) is administered to a subject intraocularly or byintraocular means. In another embodiment, a compound, molecule, orcomposition for the treatment of ocular conditions (e.g., maculardegeneration, diabetic retinopathy etc.) is administered to a subjectperiocularly or by periocular means (see for example Ahlheim et al.,International PCT publication No. WO 03/24420). In one embodiment, asiNA molecule and/or formulation or composition thereof is administeredto a subject intraocularly or by intraocular means. In anotherembodiment, a siNA molecule and/or formulation or composition thereof isadministered to a subject periocularly or by periocular means.Periocular administration generally provides a less invasive approach toadministering siNA molecules and formulation or composition thereof to asubject (see for example Ahlheim et al., International PCT publicationNo. WO 03/24420). The use of periocular administration also minimizesthe risk of retinal detachment, allows for more frequent dosing oradministration, provides a clinically relevant route of administrationfor macular degeneration and other optic conditions, and also providesthe possibility of using reservoirs (e.g., implants, pumps or otherdevices) for drug delivery. In one embodiment, siNA compounds andcompositions of the invention are administered locally, e.g., viaintraocular or periocular means, such as injection, iontophoresis (see,for example, WO 03/043689 and WO 03/030989), or implant, about every1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 weeks), alone or in combination with other compounds and/ortherapies herein. In one embodiment, siNA compounds and compositions ofthe invention are administered systemically (e.g., via intravenous,subcutaneous, intramuscular, infusion, pump, implant etc.) about every1-50 weeks (e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49,or 50 weeks), alone or in combination with other compounds and/ortherapies described herein and/or otherwise known in the art.

In one embodiment, the invention features the use of methods to deliverthe nucleic acid molecules of the instant invention to hematopoieticcells, including monocytes and lymphocytes. These methods are describedin detail by Hartmann et al., 1998, J. Pharmacol. Exp. Ther., 285(2),920-928; Kronenwett et al., 1998, Blood, 91(3), 852-862; Filion andPhillips, 1997, Biochim. Biophys. Acta., 1329(2), 345-356; Ma and Wei,1996, Leuk. Res., 20(11/12), 925-930; and Bongartz et al., 1994, NucleicAcids Research, 22(22), 4681-8. Such methods, as described above,include the use of free oligonucleitide, cationic lipid formulations,liposome formulations including pH sensitive liposomes andimmunoliposomes, and bioconjugates including oligonucleotides conjugatedto fusogenic peptides, for the transfection of hematopoietic cells witholigonucleotides.

In one embodiment, the siNA molecules and compositions of the inventionare administered to the inner ear by contacting the siNA with inner earcells, tissues, or structures such as the cochlea, under conditionssuitable for the administration. In one embodiment, the administrationcomprises methods and devices as described in U.S. Pat. Nos. 5,421,818,5,476,446, 5,474,529, 6,045,528, 6,440,102, 6,685,697, 6,120,484; and5,572,594; all incorporated by reference herein and the teachings ofSilverstein, 1999, Ear Nose Throat J., 78, 595-8, 600; and Jackson andSilverstein, 2002, Otolaryngol Clin North Am., 35, 639-53, and adaptedfor use the siNA molecules of the invention.

In one embodiment, the siNA molecules of the invention and formulationsor compositions thereof are administered directly or topically (e.g.,locally) to the dermis or follicles as is generally known in the art(see for example Brand, 2001, Cum Opin. Mol. Ther., 3, 244-8; Regnier etal., 1998, J. Drug Target, 5, 275-89; Kanikkannan, 2002, BioDrugs, 16,339-47; Wraight et al., 2001, Pharmacol. Ther., 90, 89-104; and Preatand Dujardin, 2001, STP PharmaSciences, 11, 57-68). In one embodiment,the siNA molecules of the invention and formulations or compositionsthereof are administered directly or topically using a hydroalcoholicgel formulation comprising an alcohol (e.g., ethanol or isopropanol),water, and optionally including additional agents such isopropylmyristate and carbomer 980.

In one embodiment, a siNA molecule of the invention is administerediontophoretically, for example to a particular organ or compartment(e.g., the eye, back of the eye, heart, liver, kidney, bladder,prostate, tumor, CNS etc.). Non-limiting examples of iontophoreticdelivery are described in, for example, WO 03/043689 and WO 03/030989,which are incorporated by reference in their entireties herein.

In one embodiment, siNA compounds and compositions of the invention areadministered either systemically or locally about every 1-50 weeks(e.g., about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50weeks), alone or in combination with other compounds and/or therapiesherein. In one embodiment, siNA compounds and compositions of theinvention are administered systemically (e.g., via intravenous,subcutaneous, intramuscular, infusion, pump, implant etc.) about every1-50 weeks (e.g., about every 1, 2, 3, 4, 0.5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,49, or 50 weeks), alone or in combination with other compounds and/ortherapies described herein and/or otherwise known in the art.

In one embodiment, delivery systems of the invention include, forexample, aqueous and nonaqueous gels, creams, multiple emulsions,microemulsions, liposomes, ointments, aqueous and nonaqueous solutions,lotions, aerosols, hydrocarbon bases and powders, and can containexcipients such as solubilizers, permeation enhancers (e.g., fattyacids, fatty acid esters, fatty alcohols and amino acids), andhydrophilic polymers (e.g., polycarbophil and polyvinylpyrolidone). Inone embodiment, the pharmaceutically acceptable carrier is a liposome ora transdermal enhancer. Examples of liposomes which can be used in thisinvention include the following: (1) CellFectin, 1:1.5 (M/M) liposomeformulation of the cationic lipidN,NLNILNIII-tetramethyl-N,NLNILNIII-tetrapalmit-y-spermine and dioleoylphosphatidylethanolamine (DOPE) (GIBCO BRL); (2) Cytofectin GSV, 2:1(M/M) liposome formulation of a cationic lipid and DOPE (Glen Research);(3) DOTAP(N-[1-(2,3-dioleoyloxy)-N,N,N-tri-methyl-ammoniummethylsulfate)(Boehringer Manheim); and (4) Lipofectamine, 3:1 (M/M) liposomeformulation of the polycationic lipid DOSPA and the neutral lipid DOPE(GIBCO BRL).

In one embodiment, delivery systems of the invention include patches,tablets, suppositories, pessaries, gels and creams, and can containexcipients such as solubilizers and enhancers (e.g., propylene glycol,bile salts and amino acids), and other vehicles (e.g., polyethyleneglycol, fatty acid esters and derivatives, and hydrophilic polymers suchas hydroxypropylmethylcellulose and hyaluronic acid).

In one embodiment, siNA molecules of the invention are formulated orcomplexed with polyethylenimine (e.g., linear or branched PEI) and/orpolyethylenimine derivatives, including for example grafted PEIs such asgalactose PEI, cholesterol PEI, antibody derivatized PEI, andpolyethylene glycol PEI (PEG-PEI) derivatives thereof (see for exampleOgris et al., 2001, AAPA PharmSci, 3, 1-11; Furgeson et al., 2003,Bioconjugate Chem., 14, 840-847; Kunath et al., 2002, PhramaceuticalResearch, 19, 810-817; Choi et al., 2001, Bull. Korean Chem. Soc., 22,46-52; Bettinger et al., 1999, Bioconjugate Chem., 10, 558-561; Petersonet al., 2002, Bioconjugate Chem., 13, 845-854; Erbacher et al., 1999,Journal of gene Medicine Preprint, 1, 1-18; Godbey et al., 1999., PNASUSA, 96, 5177-5181; Godbey et al., 1999, Journal of Controlled Release,60, 149-160; Diebold et al., 1999, Journal of Biological Chemistry, 274,19087-19094; Thomas and Klibanov, 2002, PNAS USA, 99, 14640-14645; andSagara, U.S. Pat. No. 6,586,524, incorporated by reference herein.

In one embodiment, a siNA molecule of the invention comprises abioconjugate, for example a nucleic acid conjugate as described inVargeese et al., U.S. Ser. No. 10/427,160, filed Apr. 30, 2003; U.S.Pat. Nos. 6,528,631; 6,335,434; 6,235,886; 6,153,737; 5,214,136;5,138,045, all incorporated by reference herein.

Thus, the invention features a pharmaceutical composition comprising oneor more nucleic acid(s) of the invention in an acceptable carrier, suchas a stabilizer, buffer, and the like. The polynucleotides of theinvention can be administered (e.g., RNA, DNA or protein) and introducedto a subject by any standard means, with or without stabilizers,buffers, and the like, to form a pharmaceutical composition. When it isdesired to use a liposome delivery mechanism, standard protocols forformation of liposomes can be followed. The compositions of the presentinvention can also be formulated and used as creams, gels, sprays, oilsand other suitable compositions for topical, dermal, or transdermaladministration as is known in the art.

The present invention also includes pharmaceutically acceptableformulations of the compounds described. These formulations includesalts of the above compounds, e.g., acid addition salts, for example,salts of hydrochloric, hycirobromic, acetic acid, and benzene sulfonicacid.

A pharmacological composition or formulation refers to a composition orformulation in a form suitable for administration, e.g., systemic orlocal administration, into a cell or subject, including for example ahuman. Suitable forms, in part, depend upon the use or the route ofentry, for example oral, transdennal, or by injection. Such forms shouldnot prevent the composition or formulation from reaching a target cell(i.e., a cell to which the negatively charged nucleic acid is desirablefor delivery). For example, pharmacological compositions injected intothe blood stream should be soluble. Other factors are known in the art,and include considerations such as toxicity and forms that prevent thecomposition or formulation from exerting its effect.

In one embodiment, siNA molecules of the invention are administered to asubject by systemic administration in a pharmaceutically acceptablecomposition or formulation. By “systemic administration” is meant invivo systemic absorption or accumulation of drugs in the blood streamfollowed by distribution throughout the entire body. Administrationroutes that lead to systemic absorption include, without limitation:intravenous, subcutaneous, portal vein, intraperitoneal, inhalation,oral, intrapulmonary and intramuscular. Each of these administrationroutes exposes the siNA molecules of the invention to an accessiblediseased tissue (e.g., lung)., The rate of entry of a drug into thecirculation has been shown to be a function of molecular weight or size.The use of a liposome or other drug carrier comprising the compounds ofthe instant invention can potentially localize the drug, for example, incertain tissue types, such as the tissues of the reticular endothelialsystem (RES). A liposome formulation that can facilitate the associationof drug with the surface of cells, such as, lymphocytes and macrophagesis also useful. This approach can provide enhanced delivery of the drugto target cells by taking advantage of the specificity of macrophage andlymphocyte immune recognition of abnormal cells.

By “pharmaceutically acceptable formulation” or “pharmaceuticallyacceptable composition” is meant, a composition or formulation thatallows for the effective distribution of the nucleic acid molecules ofthe instant invention in the physical location most suitable for theirdesired activity. Non-limiting examples of agents suitable forformulation with the nucleic acid molecules of the instant inventioninclude: P-glycoprotein inhibitors (such as Pluronic P85); biodegradablepolymers, such as poly (DL-lactide-coglycolide) microspheres forsustained release delivery (Emerich, D F et al, 1999, Cell Transplant,8, 47-58); and loaded nanoparticles, such as those made ofpolybutylcyanoacrylate. Other non-limiting examples of deliverystrategies for the nucleic acid molecules of the instant inventioninclude material described in Boado et al., 1998, J. Pharm. Sci., 87,1308-1315; Tyler et al., 1999, FEBS Lett., 421, 280-284; Pardridge etal., 1995, PNAS USA., 92, 5592-5596; Boado, 1995, Adv. Drug DeliveryRev., 15, 73-107; Aldrian-Herrada et al., 1998, Nucleic Acids Res., 26,4910-4916; and Tyler et al., 1999, PNAS USA., 96, 7053-7058.

The invention also features the use of a composition comprisingsurface-modified liposomes containing poly (ethylene glycol) lipids(PEG-modified, or long-circulating liposomes or stealth liposomes) andnucleic acid molecules of the invention. These formulations offer amethod for increasing the accumulation of drugs (e.g., siNA) in targettissues. This class of drug carriers resists opsonization andelimination by the mononuclear phagocytic system (MPS or RES), therebyenabling longer blood circulation times and enhanced tissue exposure forthe encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627;Ishiwata et al., Chem. Pharm. Butt. 1995, 43, 1005-1011). Such liposomeshave been shown to accumulate selectively in tumors, presumably byextravasation and capture in the neovascularized target tissues (Lasicet al., Science 1995, 267, 1275-1276; Oku et al., 1995, Biochim.Biophys. Acta, 1238, 86-90). The long-circulating liposomes enhance thepharmacokinetics and pharmacodynamics of DNA and RNA, particularlycompared to conventional cationic liposomes which are known toaccumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42,24864-24870; Choi et al., International PCT Publication No. WO 96/10391;Ansell et al., International PCT Publication No. WO 96/10390; Holland etal., International PCT Publication No. WO 96/10392). Long-circulatingliposomes are also likely to protect drugs from nuclease degradation toa greater extent compared to cationic liposomes, based on their abilityto avoid accumulation in metabolically aggressive MPS tissues such asthe liver and spleen.

In one embodiment, a liposomal formulation of the invention comprises adouble stranded nucleic acid molecule of the invention (e.g, siNA)formulated or complexed with compounds and compositions described inU.S. Pat. Nos. 6,858,224; 6,534,484; 6,287,591; 6,835,395; 6,586,410;6,858,225; 6,815,432; 6,586,001; 6,120,798; 6,977,223; 6,998,115;5,981,501; 5,976,567; 5,705,385; US 2006/0019912; US 2006/0019258; US2006/0008909; US 2005/0255153; US 2005/0079212; US 2005/0008689; US2003/0077829, US 2005/0064595, US 2005/0175682, US 2005/0118253; US2004/0071654; US 2005/0244504; US 2005/0265961 and US 2003/0077829, allof which are incorporated by reference herein in their entirety.

The present invention also includes compositions prepared for storage oradministration that include a pharmaceutically effective amount of thedesired compounds in a pharmaceutically acceptable carrier or diluent.Acceptable carriers or diluents for therapeutic use are well known inthe pharmaceutical art, and are described, for example, in Remington'sPharmaceutical Sciences, Mack Publishing Co. (A.R. Gennaro edit. 1985),hereby incorporated by reference herein. For example, preservatives,stabilizers, dyes and flavoring agents can be provided. These includesodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Inaddition, antioxidants and suspending agents can be used.

A pharmaceutically effective dose is that dose required to prevent,inhibit the occurrence, or treat (alleviate a symptom to some extent,preferably all of the symptoms) of a disease state. The pharmaceuticallyeffective dose depends on the type of disease, the composition used, theroute of administration, the type of mammal being treated, the physicalcharacteristics of the specific mammal under consideration, concurrentmedication, and other factors that those skilled in the medical artswill recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kgbody weight/day of active ingredients is administered dependent uponpotency of the negatively charged polymer.

The nucleic acid molecules of the invention and formulations thereof canbe administered orally, topically, parenterally, by inhalation or spray,or rectally in dosage unit formulations containing conventionalnon-toxic pharmaceutically acceptable carriers, adjuvants and/orvehicles. The term parenteral as used herein includes percutaneous,subcutaneous, intravascular (e.g., intravenous), intramuscular, orintrathecal injection or infusion techniques and the like. In addition,there is provided a pharmaceutical formulation comprising a nucleic acidmolecule of the invention and a pharmaceutically acceptable carrier. Oneor more nucleic acid molecules of the invention can be present inassociation with one or more non-toxic pharmaceutically acceptablecarriers and/or diluents and/or adjuvants, and if desired other activeingredients. The pharmaceutical compositions containing nucleic acidmolecules of the invention can be in a form suitable for oral use, forexample, as tablets, troches, lozenges, aqueous or oily suspensions,dispersible powders or granules, emulsion, hard or soft capsules, orsyrups or elixirs.

Compositions intended for oral use can be prepared according to anymethod known to the art for the manufacture of pharmaceuticalcompositions and such compositions can contain one or more suchsweetening agents, flavoring agents, coloring agents or preservativeagents in order to provide pharmaceutically elegant and palatablepreparations. Tablets contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients, that are suitable forthe manufacture of tablets. These excipients can be, for example, inertdiluents; such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia; and lubricating agents, for example magnesiumstearate, stearic acid or talc. The tablets can be uncoated or they canbe coated by known techniques. In some cases such coatings can beprepared by known techniques to delay disintegration and absorption inthe gastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonosterate or glyceryl distearate can be employed.

Formulations for oral use can also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with water or anoil medium, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in a mixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydropropyl-methylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents can be a naturally-occurring phosphatide,for example, lecithin, or condensation products of an alkylene oxidewith fatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyleneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions can also contain one or more preservatives, forexample ethyl, or n-propyl phydroxybenzoate, one or more coloringagents, one or more flavoring agents, and one or more sweetening agents,such as sucrose or saccharin.

Oily suspensions can be formulated by suspending the active ingredientsin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions can contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents and flavoring agents can beadded to provide palatable oral preparations. These compositions can bepreserved by the addition of an anti-oxidant such as ascorbic acid

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents orsuspending agents are exemplified by those already mentioned above.Additional excipients, for example sweetening, flavoring and coloringagents, can also be present.

Pharmaceutical compositions of the invention can also be in the form ofoil-in water emulsions. The oily phase can be a vegetable oil or amineral oil or mixtures of these. Suitable emulsifying agents can benaturally-occurring gums, for example gum acacia or gum tragacanth,naturally-occurring phosphatides, for example soy bean, lecithin, andesters or partial esters derived from fatty acids and hexitol,anhydrides, for example sorbitan monooleate, and condensation productsof the said partial esters with ethylene oxide, for examplepolyoxyethylene sorbitan monooleate. The emulsions can also containsweetening and flavoring agents.

Syrups and elixirs can be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol, glucose or sucrose. Suchformulations can also contain a demulcent, a preservative and flavoringand coloring agents. The pharmaceutical compositions can be in the formof a sterile injectable aqueous or oleaginous suspension. Thissuspension can be formulated according to the known art using thosesuitable dispersing or wetting agents and suspending agents that havebeen mentioned above. The sterile injectable preparation can also be asterile injectable solution or suspension in a non-toxic parentallyacceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can beemployed are water, Ringer's solution and isotonic sodium chloridesolution. In addition, sterile, fixed oils are conventionally employedas a solvent or suspending medium. For this purpose, any bland fixed oilcan be employed including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

The nucleic acid molecules of the invention can also be administered inthe form of suppositories, e.g., for rectal administration of the drug.These compositions can be prepared by mixing the drug with a suitablenon-irritating excipient that is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include cocoa butter andpolyethylene glycols.

Nucleic acid molecules of the invention can be administered parenterallyin a sterile medium. The drug, depending on the vehicle andconcentration used, can either be suspended or dissolved in the vehicle.Advantageously, adjuvants such as local anesthetics, preservatives andbuffering agents can be dissolved in the vehicle.

Dosage levels of the order of from about 0.1 mg to about 140 mg perkilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per subject perday). The amount of active ingredient that can be combined with thecarrier materials to produce a single dosage form varies depending uponthe host treated and the particular mode of administration. Dosage unitforms generally contain between from about 1 mg to about 500 mg of anactive ingredient.

It is understood that the specific dose level for any particular subjectdepends upon a variety of factors including the activity of the specificcompound employed, the age, body weight, general health, sex, diet, timeof administration, route of administration, and rate of excretion, drugcombination and the severity of the particular disease undergoingtherapy.

For administration to non-human animals, the composition can also beadded to the animal feed or drinking water. It can be convenient toformulate the animal feed and drinking water compositions so that theanimal takes in a therapeutically appropriate quantity of thecomposition along with its diet. It can also be convenient to presentthe composition as a premix for addition to the feed or drinking water.

The nucleic acid molecules of the present invention can also beadministered to a subject in combination with other therapeuticcompounds to increase the overall therapeutic effect. The use ofmultiple compounds to treat an indication can increase the beneficialeffects while reducing the presence of side effects.

In one embodiment, the invention comprises compositions suitable foradministering nucleic acid molecules of the invention to specific celltypes. For example, the asialoglycoprotein receptor (ASGPr) (Wu and Wu,1987, J Biol. Chem. 262, 4429-4432) is unique to hepatocytes and bindsbranched galactose-terminal glycoproteins, such as asialoorosomucoid(ASOR). In another example, the folate receptor is overexpressed in manycancer cells. Binding of such glycoproteins, synthetic glycoconjugates,or folates to the receptor takes place with an affinity that stronglydepends on the degree of branching of the oligosaccharide chain, forexample, triatennary structures are bound with greater affinity thanbiatenarry or monoatemiary chains (Baenziger and Fiete, 1980, Cell 22,611-620; Connolly et al., 1982, J Biol. Chem., 257, 939-945). Lee andLee, 1987, Glycoconjugate J., 4, 317-328, obtained this high specificitythrough the use of N-acetyl-D-galactosamine as the carbohydrate moiety,which has higher affinity for the receptor, compared to galactose. This“clustering effect” has also been described for the binding and uptakeof mannosyl-terminating glycoproteins or glycoconjugates (Ponpipom etal., 1981, J Med Chem., 24, 1388-1395). The use of galactose,galactosamine, or folate based conjugates to transport exogenouscompounds across cell membranes can provide a targeted delivery approachto, for example, the treatment of liver disease, cancers of the liver,or other cancers. The use of bioconjugates can also provide a reductionin the required dose of therapeutic compounds required for treatment.Furthermore, therapeutic bioavailability, pharmacodynamics, andpharmacokinetic parameters can be modulated through the use of nucleicacid bioconjugates of the invention. Non-limiting examples of suchbioconjugates are described in Vargeese et al., U.S. Ser. No.10/201,394, filed Aug. 13, 2001; and Matulic-Adamic et al., U.S. Ser.No. 60/362,016, filed Mar. 6, 2002.

Alternatively, certain siNA molecules of the instant invention can beexpressed within cells from eukaryotic promoters (e.g., Izant andWeintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc.Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad.Sci. USA, 88, 10591-5; Kashani-Sabet et al., 1992, Antisense Res. Dev.,2, 3-15; Dropulic et al., 1992, J Virol., 66, 1432-41; Weerasinghe etal., 1991, J Virol., 65, 5531-4; Ojwang et al., 1992, Proc. Natl. Acad.Sci. USA, 89, 10802-6; Chen et al., 1992, Nucleic Acids Res., 20,4581-9; Sarver et al., 1990 Science, 247, 1222-1225; Thompson et al.,1995, Nucleic Acids Res., 23, 2259; Good et al., 1997, gene Therapy, 4,45. Those skilled in the art realize that any nucleic acid can beexpressed in eukaryotic cells from the appropriate DNA/RNA vector. Theactivity of such nucleic acids can be augmented by their release fromthe primary transcript by a enzymatic nucleic acid (Draper et al., PCTWO 93/23569, and Sullivan et al., PCT WO 94/02595; Ohkawa et al., 1992,Nucleic Acids Syinp. Ser., 27, 15-6; Taira et al., 1991, Nucleic AcidsRes., 19, 5125-30; Ventura et al., 1993, Nucleic Acids Res., 21,3249-55; Chowrira et al., 1994, J. Biol. Chem., 269, 25856.

In another aspect of the invention, RNA molecules of the presentinvention can be expressed from transcription units (see for exampleCouture et al., 1996, TIG., 12, 510) inserted into DNA or RNA vectors.The recombinant vectors can be DNA plasmids or viral vectors. siNAexpressing viral vectors can be constructed based on, but not limitedto, adeno-associated virus, retrovirus, adenovirus, or alphavirus. Inanother embodiment, pol III based constructs are used to express nucleicacid molecules of the invention (see for example Thompson, U.S. Pat.Nos. 5,902,880 and 6,146,886). The recombinant vectors capable ofexpressing the siNA molecules can be delivered as described above, andpersist in target cells. Alternatively, viral vectors can be used thatprovide for transient expression of nucleic acid molecules. Such vectorscan be repeatedly administered as necessary. Once expressed, the siNAmolecule interacts with the target mRNA and generates an RNAi response.Delivery of siNA molecule expressing vectors can be systeMic, such as byintravenous or intramuscular administration, by administration to targetcells ex-planted from a subject followed by reintroduction into thesubject, or by any other means that would allow for introduction intothe desired target cell (for a review see Couture et al., 1996, TIG.,12, 510).

In one aspect the invention features an expression vector comprising anucleic acid sequence encoding at least one siNA molecule of the instantinvention. The expression vector can encode one or both strands of asiNA duplex, or a single self-complementary strand that self hybridizesinto a siNA duplex. The nucleic acid sequences encoding the siNAmolecules of the instant invention can be operably linked in a mannerthat allows expression of the siNA molecule (see for example Paul etal., 2002, Nature Biotechnology, 19, 505; Miyagishi and Taira, 2002,Nature Biotechnology, 19, 497; Lee et al., 2002, Nature Biotechnology,19, 500; and Novina et al., 2002, Nature Medicine, advance onlinepublication doi:10.1038/nm725).

In another aspect, the invention features an expression vectorcomprising: a) a transcription initiation region (e.g., eukaryotic polI, II or III initiation region); b) a transcription termination region(e.g., eukaryotic pol I, II or III termination region); and c) a nucleicacid sequence encoding at least one of the siNA molecules of the instantinvention, wherein said sequence is operably linked to said initiationregion and said termination region in a manner that allows expressionand/or delivery of the siNA molecule. The vector can optionally includean open reading frame (ORF) for a protein operably linked on the 5′ sideor the 3′-side of the sequence encoding the siNA of the invention;and/or an intron (intervening sequences).

Transcription of the siNA molecule sequences can be driven from apromoter for eukaryotic RNA polymerase I (pol I), RNA polymerase II (polII), or RNA polymerase III (pol III). Transcripts from pol II or pol IIIpromoters are expressed at high levels in all cells; the levels of agiven pol II promoter in a given cell type depends on the nature of thegene regulatory sequences (enhancers, silencers, etc.) present nearby.Prokaryotic RNA polymerase promoters are also used, providing that theprokaryotic RNA polymerase enzyme is expressed in the appropriate cells(Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci. USA, 87, 6743-7; Gaoand Huang 1993, Nucleic Acids Res., 21, 2867-72; Lieber et al., 1993,Methods Enzymol., 217, 47-66; Zhou et al., 1990, Mol. Cell. Biol., 10,4529-37). Several investigators have demonstrated that nucleic acidmolecules expressed from such promoters can function in mammalian cells(e.g., Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3-15; Ojwanget al., 1992, Proc. Natl. Acad. Sci. USA, 89, 10802-6; Chen et al.,1992, Nucleic Acids Res., 20, 4581-9; Yu et al., 1993, Proc. Natl. Acad.Sci. USA, 90, 6340-4; L'Huillier et al., 1992, EMBO 1, 11, 4411-8;Lisziewicz et al., 1993, Proc. Natl. Acad. Sci. U S. A, 90, 8000-4;Thompson et al., 1995, Nucleic Acids Res., 23, 2259; Sullenger & Cech,1993, Science, 262, 1566). More specifically, transcription units suchas the ones derived from genes encoding U6 small nuclear (snRNA),transfer RNA (tRNA) and adenovirus VA RNA are useful in generating highconcentrations of desired RNA molecules such as siNA in cells (Thompsonet al., supra; Couture and Stinchcomb, 1996, supra; Noonberg et al.,1994, Nucleic Acid Res., 22, 2830; Noonberg et al., U.S. Pat. No.5,624,803; Good et al., 1997, gene Ther., 4, 45; Beigelman et al.,International PCT Publication No. WO 96/18736. The above siNAtranscription units can be incorporated into a variety of vectors forintroduction into mammalian cells, including but not restricted to,plasmid DNA vectors, viral DNA vectors (such as adenovirus oradeno-associated virus vectors), or viral RNA vectors (such asretroviral or alphavirus vectors) (for a review see Couture andStinchcomb, 1996, supra).

In another aspect the invention features an expression vector comprisinga nucleic acid sequence encoding at least one of the siNA molecules ofthe invention in a manner that allows expression of that siNA molecule.The expression vector comprises in one embodiment; a) a transcriptioninitiation region; b) a transcription termination region; and c) anucleic acid sequence encoding at least one strand of the siNA molecule,wherein the sequence is operably linked to the initiation region and thetermination region in a manner that allows expression and/or delivery ofthe siNA molecule.

In another embodiment the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an open reading frame; and d) a nucleic acid sequence encoding atleast one strand of a siNA molecule, wherein the sequence is operablylinked to the 3′-end of the open reading frame and wherein the sequenceis operably linked to the initiation region, the open reading frame andthe termination region in a manner that allows expression and/ordelivery of the siNA molecule. In yet another embodiment, the expressionvector comprises: a) a transcription initiation region; b) atranscription termination region; c) an intron; and d) a nucleic acidsequence encoding at least one siNA molecule, wherein the sequence isoperably linked to the initiation region, the intron and the terminationregion in a manner which allows expression and/or delivery of thenucleic acid molecule.

In another embodiment, the expression vector comprises: a) atranscription initiation region; b) a transcription termination region;c) an intron; d) an open reading frame; and e) a nucleic acid sequenceencoding at least one strand of a siNA molecule, wherein the sequence isoperably linked to the 3′-end of the open reading frame and wherein thesequence is operably ‘linked to the initiation region, the intron, theopen reading frame and the termination region in a manner which allowsexpression and/or delivery of the siNA molecule.

EXAMPLES

The following are non-limiting examples showing the selection,isolation, synthesis and activity of nucleic acids of the instantinvention.

Example 1: Tandem Synthesis of siNA Constructs

Exemplary siNA molecules of the invention are synthesized in tandemusing a cleavable linker, for example, a succinyl-based linker. Tandemsynthesis as described herein is followed by a one-step purificationprocess that provides RNAi molecules in high yield. This approach ishighly amenable to siNA synthesis in support of high throughput RNAiscreening, and can be readily adapted to multi-column or multi-wellsynthesis platforms.

After completing a tandem synthesis of a siNA oligo and its complementin which the 5′-terminal dimethoxytrityl (5′-O-DMT) group remains intact(trityl on synthesis), the oligonucleotides are deprotected as describedabove. Following deprotection, the siNA sequence strands are allowed tospontaneously hybridize. This hybridization yields a duplex in which onestrand has retained the 5′-O-DMT group while the complementary strandcomprises a terminal 5′-hydroxyl. The newly formed duplex behaves as asingle molecule during routine solid-phase extraction purification(Trityl-On purification) even though only one molecule has adimethoxytrityl group. Because the strands form a stable duplex, thisdimethoxytrityl group (or an equivalent group, such as other tritylgroups or other hydrophobic moieties) is all that is required to purifythe pair of oligos, for example, by using a C18 cartridge.

Standard phosphoramidite synthesis chemistry is used up to the point ofintroducing a tandem linker, such as an inverted deoxy abasic succinateor glyceryl succinate linker (see FIG. 1) or an equivalent cleavablelinker. A non-limiting example of linker coupling conditions that can beused includes a hindered base such as diisopropylethylamine (DIPA)and/or DMAP in the presence of an activator reagent such asBromotripyrrolidinophosphoniumhexaflurorophosphate (PyBrOP). After thelinker is coupled, standard synthesis chemistry is utilized to completesynthesis of the second sequence leaving the terminal the 5′-O-DMTintact. Following synthesis, the resulting oligonucleotide isdeprotected according to the procedures described herein and quenchedwith a suitable buffer, for example with 50 mM NaOAc or 1.5M NH₄H₂CO₃.

Purification of the siNA duplex can be readily accomplished using solidphase extraction, for example, using a Waters C18 SepPak 1 g cartridgeconditioned with 1 column volume (CV) of acetonitrile, 2 CV H2O, and 2CV 50 mM NaOAc. The sample is loaded and then washed with 1 CV H₂O or 50mM NaOAc. Failure sequences are eluted with 1 CV 14% ACN (Aqueous with50 mM NaOAc and 50 mM NaCl). The column is then washed, for example with1 CV H₂O followed by on-column detritylation, for example by passing 1CV of 1% aqueous trifluoroacetic acid (TFA) over the column, then addinga second CV of 1% aqueous TFA to the column and allowing to stand forapproximately 10 minutes. The remaining TFA solution is removed and thecolumn washed with H₂O followed by 1 CV 1M NaCl and additional H2O. ThesiNA duplex product is then eluted, for example, using 1 CV 20% aqueousCAN.

FIG. 2 provides an example of MALDI-TOF mass spectrometry analysis of apurified siNA construct in which each peak corresponds to the calculatedmass of an individual siNA strand of the siNA duplex. The same purifiedsiNA provides three peaks when analyzed by capillary gel electrophoresis(CGE), one peak presumably corresponding to the duplex siNA, and twopeaks presumably corresponding to the separate siNA sequence strands.Ion exchange HPLC analysis of the same siNA contract only shows a singlepeak. Testing of the purified siNA construct using a luciferase reporterassay described below demonstrated the same RNAi activity compared tosiNA constructs generated from separately synthesized oligonucleotidesequence strands.

Example 2: Identification of Potential siNA Target Sites in any RNASequence

The sequence of an RNA target of interest, such as a human mRNAtranscript (e.g., any of sequences referred to herein by GenBankAccession Number), is screened for target sites, for example by using acomputer folding algorithm. In a non-limiting example, the sequence of agene or RNA gene transcript derived from a database, such as Genbank, isused to generate siNA targets having complementarity to the target. Suchsequences can be obtained from a database, or can be determinedexperimentally as known in the art. Target sites that are known, forexample, those target sites determined to be effective target sitesbased on studies with other nucleic acid molecules, for exampleribozymes or antisense, or those targets known to be associated with adisease, trait, or condition such as those sites containing mutations ordeletions, can be used to design siNA molecules targeting those sites.Various parameters can be used to determine which sites are the mostsuitable target sites within the target RNA sequence. These parametersinclude but are not limited to secondary or tertiary RNA structure, thenucleotide base composition of the target sequence, the degree ofhomology between various regions of the target sequence, or the relativeposition of the target sequence within the RNA transcript. Based onthese determinations, any number of target sites within the RNAtranscript can be chosen to screen siNA molecules for efficacy, forexample by using in vitro RNA cleavage assays, cell culture, or animalmodels. In a non-limiting example, anywhere from 1 to 1000 target sitesare chosen within the transcript based on the size of the siNA constructto be used. High throughput screening assays can be developed forscreening siNA molecules using methods known in the art, such as withmulti-well or multi-plate assays to determine efficient reduction intarget gene expression.

Example 3: Selection of siNA Molecule Target Sites in a RNA

The following non-limiting steps can be used to carry out the selectionof siNAs targeting a given gene sequence or transcript.

1. The target sequence is parsed in silico into a list of all fragmentsor subsequences of a particular length, for example 23 nucleotidefragments, contained within the target sequence. This step is typicallycarried out using a custom Perl script, but commercial sequence analysisprograms such as Oligo, MacVector, or the GCG Wisconsin Package can beemployed as well.

2. In some instances the siNAs correspond to more than one targetsequence; such would be the case for example in targeting differenttranscripts of the same gene, targeting different transcripts of morethan one gene, or for targeting both the human gene and an animalhomolog. In this case, a subsequence list of a particular length isgenerated for each of the targets, and then the lists are compared tofind matching sequences in each list. The subsequences are then rankedaccording to the number of target sequences that contain the givensubsequence; the goal is to find subsequences that are present in mostor all of the target sequences. Alternately, the ranking can identifysubsequences that are unique to a target sequence, such as a mutanttarget sequence. Such an approach would enable the use of siNA to targetspecifically the mutant sequence and not effect the expression of thenormal sequence.

3. In some instances the siNA subsequences are absent in one or moresequences while present in the desired target sequence; such would bethe case if the siNA targets a gene with a paralogous family member thatis to remain untargeted. As in Step 2 above, a subsequence list of aparticular length is generated for each of the targets, and then thelists are compared to find sequences that are present in the target genebut are absent in the untargeted paralog.

4. The ranked siNA subsequences can be further analyzed and rankedaccording to GC content. A preference can be given to sites containing30-70% GC, with a further preference to sites containing 40-60% GC.

5. The ranked siNA subsequences can be further analyzed and rankedaccording to self-folding and internal hairpins. Weaker internal foldsare preferred; strong hairpin structures are to be avoided.

6. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have runs of GGG or CCC in the sequence. GGG(or even more Gs) in either strand can make oligonucleotide synthesisproblematic and can potentially interfere with RNAi activity, so it isavoided whenever better sequences are available. CCC is searched in thetarget strand because that will place GGG in the antisense strand.

7. The ranked siNA subsequences can be further analyzed and rankedaccording to whether they have the dinucleotide UU (uridinedinucleotide) on the 3′-end of the sequence, and/or AA on the 5′-end ofthe sequence (to yield 3′ UU on the antisense sequence). These sequencesallow one to design siNA molecules with terminal TT thymidinedinucleotides.

8. Four or five target sites are chosen from the ranked list ofsubsequences as described above. For example, in subsequences having 23nucleotides, the right 21 nucleotides of each chosen 23-mer subsequenceare then designed and synthesized for the upper (sense) strand of thesiNA duplex, while the reverse complement of the left 21 nucleotides ofeach chosen 23-mer subsequence are then designed and synthesized for thelower (antisense) strand of the siNA duplex (see Table II). If terminalTT residues are desired for the sequence (as described in Step 7 above),then the two 3′ terminal nucleotides of both the sense and antisensestrands are replaced by TT prior to synthesizing the oligos.

9. The siNA molecules are screened in an in vitro, cell culture oranimal model system to identify the most active siNA molecule or themost preferred target site within the target RNA sequence.

10. Other design considerations can be used when selecting targetnucleic acid sequences, see, for example, Reynolds et al., 2004, NatureBiotechnology Advanced Online Publication, 1 Feb. 2004,doi:10.1038/nbt936 and Ui-Tei et al., 2004, Nucleic Acids Research, 32,doi:10.1093/nar/gkh247.

In an alternate approach, a pool of siNA constructs specific to a targetsequence is used to screen for target sites in cells expressing targetRNA, such as cultured Jurkat, HeLa, A549 or 293T cells. The generalstrategy used in this approach is shown in FIG. 9. Cells expressing thetarget RNA are transfected with the pool of siNA constructs and cellsthat demonstrate a phenotype associated with target inhibition aresorted. The pool of siNA constructs can be expressed from transcriptioncassettes inserted into appropriate vectors (see for example FIG. 7 andFIG. 8). The siNA from cells demonstrating a positive phenotypic change(e.g., decreased proliferation, decreased target mRNA levels ordecreased target protein expression), are sequenced to determine themost suitable target site(s) within the target RNA sequence.

In one embodiment, siNA molecules of the invention are selected usingthe following methodology. The following guidelines were compiled topredict hyper-active siNAs that contain chemical modifications describedherein. These rules emerged from a comparative analysis of hyper-active(>75% knockdown of target mRNA levels) and inactive (<75% knockdown oftarget mRNA levels) siNAs against several different targets. A total of242 siNA sequences were analyzed. Thirty-five siNAs out of 242 siNAswere grouped into hyper-active and the remaining siNAs were grouped intoinactive groups. The hyper-active siNAs clearly showed a preference forcertain bases at particular nucleotide positions within the siNAsequence. For example, A or U nucleobase was overwhelmingly present atposition 19 of the sense strand in hyper-active siNAs and opposite wastrue for inactive siNAs. There was also a pattern of a A/U rich (3 outof 5 bases as A or U) region between positions 15-19 and G/C rich regionbetween positions 1-5 (3 out of 5 bases as G or C) of the sense strandin hyperactive siNAs. As shown in Table V, 12 such patterns wereidentified that were characteristics of hyper-active siNAs. It is to benoted that not every pattern was present in each hyper-active siNA.Thus, to design an algorithm for predicting hyper-active siNAs, adifferent score was assigned for each pattern. Depending on howfrequently such patterns occur in hyper-active siNAs versus inactivesiNAs, the design parameters were assigned a score with the highestbeing 10. If a certain nucleobase is not preferred at a position, then anegative score was assigned. For example, at positions 9 and 13 of thesense strand, a G nucleotide was not preferred in hyper-active siNAs andtherefore they were given score of −3 (minus 3). The differential scorefor each pattern is given in Table V. The pattern #4 was given a maximumscore of −100. This is mainly to weed out any sequence that containsstring of 4Gs or 4Cs as they can be highly incompatible for synthesisand can allow sequences to self-aggregate, thus rendering the siNAinactive. Using this algorithm, the highest score possible for any siNAis 66. As there are numerous siNA sequences possible against any giventarget of reasonable size (˜1000 nucleotides), this algorithm is usefulto generate hyper-active siNAs.

In one embodiment, rules 1-11 shown in Table V are used to generateactive siNA molecules of the invention. In another embodiment, rules1-12 shown in Table V are used to generate active siNA molecules of theinvention.

Example 4: siNA Design

siNA target sites were chosen by analyzing sequences of the target andoptionally prioritizing the target sites on the basis of the rulespresented in Example 3 above, and alternately on the basis of folding(structure of any given sequence analyzed to determine siNAaccessibility to the target), or by using a library of siNA molecules asdescribed in Example 3, or alternately by using an in vitro siNA systemas described in Example 6 herein. siNA molecules were designed thatcould bind each target and are selected using the algorithm above andare optionally individually analyzed by computer folding to assesswhether the siNA molecule can interact with the target sequence. Varyingthe length of the siNA molecules can be chosen to optimize activity.Generally, a sufficient number of complementary nucleotide bases arechosen to bind to, or otherwise interact with, the target RNA, but thedegree of complementarity can be modulated to accommodate siNA duplexesor varying length or base composition. By using such methodologies, siNAmolecules can be designed to target sites within any known RNA sequence,for example those RNA sequences corresponding to the any genetranscript.

Target sequences are analysed to generate targets from which doublestranded siNA are designed (Table II). To generate synthetic siNAconstructs, the algorithm described in Example 3 is utilized to pickactive double stranded constructs and chemically modified versionsthereof. For example, in Table II, the target sequence is shown, alongwith the upper (sense strand) and lower (antisense strand) of the siNAduplex. Multifunctional siNAs are designed by searching for homologoussites between different target sequences (e.g., from about 5 to about 15nucleotide regions of shared homology) and allowing for non-canonicalbase pairs (e.g., G:U wobble base pairing) or mismatched base pairs.

Chemically modified siNA constructs were designed as described herein(see for example Table I) to provide nuclease stability for systemicadministration in vivo and/or improved pharmacokinetic, localization,and delivery properties while preserving the ability to mediate RNAiactivity. Chemical modifications as described herein are introducedsynthetically using synthetic methods described herein and thosegenerally known in the art. The synthetic siNA constructs are thenassayed for nuclease stability in serum and/or cellular/tissue extracts(e.g., liver extracts). The synthetic siNA constructs are also tested inparallel for RNAi activity using an appropriate assay, such as aluciferase reporter assay as described herein or another suitable assaythat can quantity RNAi activity. Synthetic siNA constructs that possessboth nuclease stability and RNAi activity can be further modified andre-evaluated in stability and activity assays. The chemicalmodifications of the stabilized active siNA constructs can then beapplied to any siNA sequence targeting any chosen RNA and used, forexample, in target screening assays to pick lead siNA compounds fortherapeutic development (see for example FIG. 11).

Example 5: Chemical Synthesis and Purification of siNA

siNA molecules can be designed to interact with various sites in the RNAmessage, for example, target sequences within the RNA sequencesdescribed herein. The sequence of one strand of the siNA molecule(s) iscomplementary to the target site sequences described above. The siNAmolecules can be chemically synthesized using methods described herein.Inactive siNA molecules that are used as control sequences can besynthesized by scrambling the sequence of the siNA molecules such thatit is not complementary to the target sequence. Generally, siNAconstructs can by synthesized using solid phase oligonucleotidesynthesis methods as described herein (see for example Usman et al.,U.S. Pat. Nos. 5,804,683; 5,831,071; 5,998,203; 6,117,657; 6,353,098;6,362,323; 6,437,117; 6,469,158; Scaringe et al., U.S. Pat. Nos.6,111,086; 6,008,400; 6,111,086 all incorporated by reference herein intheir entirety).

In a non-limiting example, RNA oligonucleotides are synthesized in astepwisefashion using the phosphoramidite chemistry as is known in theart. Standard phosphoramidite chemistry involves the use of nucleosidescomprising any of 5′-O-dimethoxytrityl, 2′-O-tert-butyldimethylsilyl,3′-O-2-Cyanoethyl N,N-diisopropylphos-phoroamidite groups, and exocyclicamine protecting groups (e.g., N6-benzoyl adenosine, N4 acetyl cytidine,and N2-isobutyryl guanosine). Alternately, 2 r-O-Silyl Ethers can beused in conjunction with acid-labile 2′-O-orthoester protecting groupsin the synthesis of RNA as described by Scaringe supra. Differing 2′chemistries can require different protecting groups, for example2′-deoxy-2′-amino nucleosides can utilize N-phthaloyl protection asdescribed by Usman et al., U.S. Pat. No. 5,631,360, incorporated byreference herein in its entirety).

During solid phase synthesis, each nucleotide is added sequentially (3′-to 5′ direction) to the solid support-bound oligonucleotide. The firstnucleoside at the 3′-end of the chain is covalently attached to a solidsupport (e.g., controlled pore glass or polystyrene) using variouslinkers. The nucleotide precursor, a ribonucleoside phosphoramidite, andactivator are combined resulting in the coupling of the secondnucleoside phosphoramidite onto the 5′-end of the first nucleoside. Thesupport is then washed and any unreacted 5′-hydroxyl groups are cappedwith a capping reagent such as acetic anhydride to yield inactive5′-acetyl moieties. The trivalent phosphorus linkage is then oxidized toa more stable phosphate linkage. At the end of the nucleotide additioncycle, the 5′-O-protecting group is cleaved under suitable conditions(e.g., acidic conditions for trityl-based groups and fluoride forsilyl-based groups). The cycle is repeated for each subsequentnucleotide.

Modification of synthesis conditions can be used to optimize couplingefficiency, for example by using differing coupling times, differingreagent/phosphoramidite concentrations, differing contact times,differing solid supports and solid support linker chemistries dependingon the particular chemical composition of the siNA to be synthesized.Deprotection and purification of the siNA can be performed as isgenerally described in Usman et al., U.S. Pat. Nos. 5,831,071,6,353,098, 6,437,117, and Bellon et al., U.S. Pat. Nos. 6,054,576,6,162,909, 6,303,773, or Scaringe supra, incorporated by referenceherein in their entireties. Additionally, deprotection conditions can bemodified to provide the best possible yield and purity of siNAconstructs. For example, applicant has observed that oligonucleotidescomprising 2′-deoxy-2′-fluoro nucleotides can degrade underinappropriate deprotection conditions. Such oligonucleotides aredeprotected using aqueous methylamine at about 35° C. for 30 minutes. Ifthe 2′-deoxy-2′-fluoro containing oligonucleotide also comprisesribonucleotides, after deprotection with aqueous methylamine at about35° C. for 30 minutes, TEA-HF is added and the reaction maintained atabout 65° C. for an additional 15 minutes. The deprotected singlestrands of siNA are purified by anion exchange to achieve a high puritywhile maintaining high yields. To form the siNA duplex molecule thesingle strands are combined in equal molar ratios in a saline solutionto form the duplex. The duplex siNA is concentrated and desalted bytangential filtration prior to lyophilization

Example 6: RNAi In Vitro Assay to Assess siNA Activity

An in vitro assay that recapitulates RNAi in a cell-free system is usedto evaluate siNA constructs targeting target RNA targets. The assaycomprises the system described by Tuschl et al., 1999, genes andDevelopment, 13, 3191-3197 and Zamore et al., 2000, Cell, 101, 25-33adapted for use with a target RNA. A Drosophila extract derived fromsyncytial blastoderm is used to reconstitute RNAi activity in vitro.Target RNA is generated via in vitro transcription from an appropriatetarget expressing plasmid using T7 RNA polymerase or via chemicalsynthesis as described herein. Sense and antisense siNA strands (forexample 20 uM each) are annealed by incubation in buffer (such as 100 mMpotassium acetate, 30 mM HEPES-KOH, pH 7.4, 2 mM magnesium acetate) for1 minute at 90° C. followed by 1 hour at 37° C., then diluted in lysisbuffer (for example 100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4,2 mM magnesium acetate). Annealing can be monitored by gelelectrophoresis on an agarose gel in TBE buffer and stained withethidium bromide. The Drosophila lysate is prepared using zero totwo-hour-old embryos from Oregon R flies collected on yeasted molassesagar that are dechorionated and lysed. The lysate is centrifuged and thesupernatant isolated. The assay comprises a reaction mixture containing50% lysate [vol/vol], RNA (10-50 pM final concentration), and 10%[vol/vol] lysis buffer containing siNA (10 nM final concentration). Thereaction mixture also contains 10 mM creatine phosphate, 10 ug/mlcreatine phosphokinase, 100 um GTP, 100 uM UTP, 100 uM CTP, 500 uM ATP,5 mM DTT, 0.1 U/uL RNasin (Promega), and 100 uM of each amino acId. Thefinal concentration of potassium acetate is adjusted to 100 mM. Thereactions are pre-assembled on ice and preincubated at 25° C. for 10minutes before adding RNA, then incubated at 25° C. for an additional 60minutes. Reactions are quenched with 4 volumes of 1.25× Passive LysisBuffer (Promega). Target RNA cleavage is assayed by RT-PCR analysis orother methods known in the art and are compared to control reactions inwhich siNA is omitted from the reaction.

Alternately, internally-labeled target RNA for the assay is prepared byin vitro transcription in the presence of [alpha-³²p] CTP, passed over aG50 Sephadex column by spin chromatography and used as target RNAwithout further purification. Optionally, target RNA is 5′-³²P-endlabeled using T4 polynucleotide kinase enzyme. Assays are performed asdescribed above and target RNA and the specific RNA cleavage productsgenerated by RNAi are visualized on an autoradiograph of a gel. Thepercentage of cleavage is determined by PHOSPHOR IMAGER®(autoradiography) quantitation of bands representing intact control RNAor RNA from control reactions without siNA and the cleavage productsgenerated by the assay.

In one embodiment, this assay is used to determine target sites in thetarget RNA target for siNA mediated RNAi cleavage, wherein a pluralityof siNA constructs are screened for RNAi mediated cleavage of the targetRNA target, for example, by analyzing the assay reaction byelectrophoresis of labeled target RNA, or by northern blotting, as wellas by other methodology well known in the art.

Example 7: Nucleic Acid Inhibition of Target RNA

siNA molecules targeted to target RNA are designed and synthesized asdescribed above. These nucleic acid molecules can be tested for cleavageactivity in vivo, for example, using the following procedure. The targetsequences and the nucleotide location within the target RNA are given inTable II.

Two formats are used to test the efficacy of siNAs targeting any targetsequence. First, the reagents are tested in cell culture using HepG2,Jurkat, HeLa, A549 or 293T cells, to determine the extent of RNA andprotein inhibition. siNA reagents are selected against the target asdescribed herein. RNA inhibition is measured after delivery of thesereagents by a suitable transfection agent to, for example, HepG2,Jurkat, HeLa, A549 or 293T cells. Relative amounts of target RNA aremeasured versus actin using real-time PCR monitoring of amplification(e.g., ABI 7700 TAQMAN®). A comparison is made to a mixture ofoligonucleotide sequences made to unrelated targets or to a randomizedsiNA control with the same overall length and chemistry, but randomlysubstituted at each position. Primary and secondary lead reagents arechosen for the target and optimization performed. After an optimaltransfection agent concentration is chosen, a RNA time-course ofinhibition is performed with the lead siNA molecule. In addition, acell-plating format can be used to determine RNA inhibition.

Delivery of siNA to Cells

Cells (e.g., HepG2, Jurkat, HeLa, A549 or 293T cells) are seeded, forexample, at 1×10⁵ cells per well of a six-well dish in EGM-2(BioWhittaker) the day before transfection. siNA (final concentration,for example 20 nM) and cationic lipid (e.g., LNP formulations herein, oranother suitable lipid such as Lipofectamine, final concentration 2μg/ml) are complexed in EGM basal media (Biowhittaker) at 37° C. for 30minutes in polystyrene tubes. Following vortexing, the complexed siNA isadded to each well and incubated for the times indicated. For initialoptimization experiments, cells are seeded, for example, at 1×103 in 96well plates and siNA complex added as described. Efficiency of deliveryof siNA to cells is determined using a fluorescent siNA complexed withlipid. Cells in 6-well dishes are incubated with siNA for 24 hours,rinsed with PBS and fixed in 2% paraformaldehyde for 15 minutes at roomtemperature. Uptake of siNA is visualized using a fluorescentmicroscope.

TAQMAN® (Real-Time PCR Monitoring of Amplification) and LightcyclerQuantification of mRNA

Total RNA is prepared from cells following siNA delivery, for example,using Qiagen RNA purification kits for 6-well or Rneasy extraction kitsfor 96-well assays. For TAQMAN® analysis (real-time PCR monitoring ofamplification), dual-labeled probes are synthesized with the reporterdye, FAM or JOE, covalently linked at the 5′-end and the quencher dyeTAMRA conjugated to the 3′-end. One-step RT-PCR amplifications areperformed on, for example, an ABI PRISM 7700 Sequence Detector using 50μl reactions consisting of 10 μl total RNA, 100 nM forward primer, 900nM reverse primer, 100 nM probe, 1×TaqMan PCR reaction buffer(PE-Applied Biosystems), 5.5 mM MgCl₂, 300 μM each dATP, dCTP, dGTP, anddTTP, 10 U RNase Inhibitor (Promega), 1.25 U AMPLITAQ GOLD® (DNApolymerase) (PE-Applied Biosystems) and 10 U M-MLV Reverse Transcriptase(Promega). The thermal cycling conditions can consist of 30 minutes at48° C., 10 minutes at 95° C., followed by 40 cycles of 15 seconds at 95°C. and 1 minute at 60° C. Quantitation of mRNA levels is determinedrelative to standards generated from serially diluted total cellular RNA(300, 100, 33, 11 ng/reaction) and normalizing to β-actin or GAPDH mRNAin parallel TAQMAN® reactions (real-time PCR monitoring ofamplification). For each gene of interest an upper and lower primer anda fluorescently labeled probe are designed. Real time incorporation ofSYBR Green I dye into a specific PCR product can be measured in glasscapillary tubes using a lightcyler. A standard curve is generated foreach primer pair using control cRNA. Values are represented as relativeexpression to GAPDH in each sample.

Western Blotting

Nuclear extracts can be prepared using a standard micro preparationtechnique (see for example Andrews and Faller, 1991, Nucleic AcidsResearch, 19, 2499). Protein extracts from supernatants are prepared,for example using TCA precipitation. An equal volume of 20% TCA is addedto the cell supernatant, incubated on ice for 1 hour and pelleted bycentrifugation for 5 minutes. Pellets are washed in acetone, dried andresuspended in water. Cellular protein extracts are run on a 10%Bis-Tris NuPage (nuclear extracts) or 4-12% Tris-Glycine (supernatantextracts) polyacrylamide gel and transferred onto nitro-cellulosemembranes. Non-specific binding can be blocked by incubation, forexample, with 5% non-fat milk for 1 hour followed by primary antibodyfor 16 hour at 4° C. Following washes, the secondary antibody isapplied, for example (1:10,000 dilution) for 1 hour at room temperatureand the signal detected with SuperSignal reagent (Pierce).

Example 8: Models Useful to Evaluate the Down-Regulation of Target GeneExpression

Evaluating the efficacy of siNA molecules of the invention in animalmodels is an important prerequisite to human clinical trials. Variousanimal models of cancer, proliferative, inflammatory, autoimmune,neurologic, ocular, respiratory, metabolic, auditory, dermatologic etc.diseases, conditions, or disorders as are known in the art can beadapted for use for preclinical evaluation of the efficacy of nucleicacid compositions of the invention in modulating target gene expressiontoward therapeutic, cosmetic, or research use.

Example 9: RNAi Mediated Inhibition of Target Gene Expression

In Vitro siNA Mediated Inhibition of Target RNA

siNA constructs (are tested for efficacy in reducing target RNAexpression in cells, (e.g., HEKn/HEKa, HeLa, A549, A375 cells). Cellsare plated approximately 24 hours before transfection in 96-well platesat 5,000-7,500 cells/well, 100 μl/well, such that at the time oftransfection cells are 70-90% confluent. For transfection, annealedsiNAs are mixed with the transfection reagent (Lipofectamine 2000,Invitrogen) in a volume of 50 μl/well and incubated for 20 minutes atroom temperature. The siNA transfection mixtures are added to cells togive a final siNA concentration of 25 nM in a volume of 150 μl. EachsiNA transfection mixture is added to 3 wells for triplicate siNAtreatments. Cells are incubated at 37° for 24 hours in the continuedpresence of the siNA transfection mixture. At 24 hours, RNA is preparedfrom each well of treated cells. The supernatants with the transfectionmixtures are first removed and discarded, then the cells are lysed andRNA prepared from each well. Target gene expression following treatmentis evaluated by RT-PCR for the target gene and for a control gene (36B4,an RNA polymerase subunit) for normalization. The triplicate data isaveraged and the standard deviations determined for each treatment.Normalized data are graphed and the percent reduction of target mRNA byactive siNAs in comparison to their respective inverted control siNAs isdetermined.

Example 10: Efficacy of Stabilized siNA Constructs with One or MoreRibonucleotides at Select Positions

Chimeric siNA constructs (see Table II) were generated that contained 6ribonucleotide blocks either on the sense strand (passenger strand) oron the antisense (guide) strand while keeping all other nucleotideschemically modified. Target HBV message knock down was observed bymeasuring protein (HBsAg) levels instead of mRNA levels. The presence ofa block of ribonucleotides at the 5′- or 3′-end of either the sensestrand or guide strand of the siNAs showed strong silencing activity, asdid the siNA constructs where the ribonucleotide block at the ends alsohad a ribonucleotide counterpart at the opposite strand. Data for HBVsite 262 siNA constructs are shown in FIG. 30, site 263 siNA constructsin FIG. 31, and site 1583 siNA constructs in FIG. 32.

Determination of 1050 values in tissue culture revealed that chimericsiNAs containing a block of 6 ribonucleotides at the terminal positionsof siNA for HBV sites 262, 263 and 1583 (see FIGS. 33 and 34) retainedactivity. Additional constructs, where the ribonucleotide content wasreduced to a single ribonucleotide residue at the 5′ terminal positionof the guide strand sequence complementary to HBV site 263 wereevaluated for their ability to mediate RNAi. In vitro experimentsrevealed that a single ribonucleotide residue at the terminal 5′position of guide strand retained the activity of a chemically modifiedsiNA duplex (7/23, 7/24 and/or 7/28 chemistry, see FIG. 35). Because ansiNA duplex containing a single ribonucleotide residue at the 5′terminal nucleotide position of the antisense strand could cleave thetarget RNA in a catalytic manner, it can be further inferred that 2′-OHgroup within the siRNA molecule do not directly participate in thecatalytic cleavage of target RNA. Additional siNA constructs designatedas Stab 7/23, 7/24, 7/25, 0.7/26, 7/27 and 7/28 stabilizationchemistries (see Table I) were evaluated for their ability to mediateRNAi. In vitro serum stability of the 7/25 siNA construct revealed thatthis construct has a half-life of >24 h in human serum.

Applicant carried out in vitro RNAi cleavage assays using HeLa celllysate as a source of RISC proteins to evaluate various siNA constructsfor their ability to induce cleavage of target RNA. Anti-HCV siNAconstructs targeting site 304 in stab 7/8 siNA configuration wereevaluated in the in vitro RNAi cleavage assay (see Table II).Site-specific cleavage of a target RNA 10 nts from the 5′ end of theguide strand sequence is diagnostic of RISC-mediated cleavage. Indeed,the site specific cleavage of target RNA at the expected position wasobserved with anti-HCV siNA targeting site 304 in stab 7/8 siRNAconfiguration. This shows that fully modified siRNA works through RNAimechanism and that presence of 2′-OH group within the siNA is notrequired for RNAi-mediated cleavage of target RNA and that 2′-OH groupwithin the siNA does not participate in the target RNA cleavage.

As described above, the presence of ribonucleotide residues at the 5′terminal nucleotide positions of the guide strand resulted in siRNAswith robust activity. The activity of siRNA constructs in which thefirst three nucleotides of the guide strand comprising2′-deoxy-2′-fluoro pyrimidines and purine ribonucleotides was evaluated.This stabilization chemistry is termed as Stab 29 (Table I). The siNAsworked equally well in both Stab 7/25 as well as Stab 7/29 chemistries(see FIG. 36). Thus, purine residues when present at the 5′ terminalnucleotide positions can be maintained as ribonucleotides in the guidestrand and the pyrimidines nucleotides in the guide strand can bechemically modified while maintaining robust RNAi activity. To establishthat these siNAs also work through RISC-mediated specific RNAdegradation, an in vitro RNAi assay using HeLa cell lysate was used.Site-specific cleavage of the target RNA 10 nts from the 5′ end of theguide strand is diagnostic of RISC-mediated cleavage. Indeed, the sitespecific cleavage of target RNA at the expected position was observedwith all three siNAs in Stab 7/25 as well as 7/25 configurations. Thissuggests that these siNA constructs work through an RNAi mechanism.

Materials and Methods

Oligonucleotide Synthesis and Characterization

siNA oligonucleotides were synthesized, deprotected and purified asdescribed herein. The integrity and purity of the final compounds wereconfirmed by standard HPLC, CE and MALDI-TOF MS methodologies.

siRNA Annealing

siNA strands (20 μM each strand) were annealed m 100 mM potassiumacetate, 30 mM HEPES-KOH at pH 7.4, 2 mM magnesium acetate. Theannealing mixture was first heated to 90° C. for 1 min and thentransferred to 37° C. for 60 mins. Annealing was confirmed bynon-denaturing PAGE and Tm assessment in 150 mM NaCl.

Serum Stability Assay

Oligonucleotides were designed such that standard ligation methods wouldgenerate full length sense or antisense strands. Prior to ligation,standard kinase methods were used with [γ-32P]ATP to generate aninternal 32P label. Ligated material was gel purified using denaturingPAGE. Trace internally-labeled sense (or antisense) was added tounlabeled material to achieve a final concentration of 20 μM. Theunlabeled complementary strand was present at 35 μM. Annealing wasperformed as described above. Duplex formation was confirmed byunmodified PAGE and subsequent visualization on a Molecular Dynamics(Sunnyvale, Calif.) Phosphoimager.

Internally-labeled, duplexed or single-stranded siRNA was added to humanserum to achieve final concentrations of 90% serum (Sigma, St. Louis,Mo.) and 2 μM siRNA duplex with a 1.5 μM excess of the unlabeledsingle-stranded siRNA. Samples were incubated at 37° C. Aliquots wereremoved at specified time points and quenched using a five secondProteinase K (20 ug) digestion (Amersham, Piscataway, N.J.) in 50 mMTris-HCl pH 7.8, 2.5 mM EDTA, 2.5% SDS, followed by addition of a 6×volume of formamide loading buffer (90% formamide, 50 mM EDTA, 0.015%xylene cyanol and bromophenol blue, 20 μM unlabeled chase oligonucletideof the same sequence as the radiolabeled strand). Samples were separatedby denaturing PAGE and visualized on a Molecular Dynamics Phosphoimager.ImageQuant (Molecular Dynamics) software was used for quantitation.

Cell Culture Studies

The human hepatoblastoma cell lines Hep G2 was grown in minimalessential Eagle media supplemented with 10% fetal calf serum, 2 mMglutamine, 0.1 mM nonessential amino acids, 1 mM sodium pyruvate, and 25mM Hepes. Replication competent cDNA was generated by excising andre-ligating the HBV genomic sequences from the psHBV-1 vector. Hep G2cells were plated (3×104 cells/well) in 96-well microtiter plates andincubated overnight. A cationic lipid/DNA/siRNA complex was formedcontaining (at final concentrations) cationic lipid (11-15 μg/mL),re-ligated psHBV-1 (4.5 μg/mL) and siRNA (25 nM) in growth Media.Following an 15 min incubation at 37° C., 20 μL of the complex was addedto the plated Hep G2 cells in 80 of growth media minus antibiotics. Themedia was removed from the cells 72 hr post-transfection for HBsAganalysis. All transfections were performed in triplicate.

HBsAg ELISA Assay

Levels of HBsAg were determined using the Genetic Systems/Bio-Rad(Richmond, Va.) HBsAg ELISA kit, as per the manufacturer's instructions.The absorbance of cells not transfected with the HBV vector was used asbackground for the assay, and thus subtracted from the experimentalsample values.

Example 12: Efficacy of Formulated siNA Constructs with DifferentOverhang Chemistries in a Chronic Model of HBV Infection

To assess the activity of chemically stabilized siNA nanoparticle (seeVargeese et al., U.S. Provisional patent application No. 60/678,531 andin related U.S. Provisional patent application No. 60/703,946, filedJul. 29, 2005, and U.S. Provisional patent application No. 60/737,024,filed Nov. 15, 2005, all incorporated by reference herein) compositionsagainst HBV, systemic dosing of the formulated siNA composition(Formulation L-086 and L-061, see Table IV and U.S. Provisional patentapplication No. 60/737,024, filed Nov. 15, 2005) was performed followinghydrodynamic injection (HDI) of the HBV vector in mouse strainNOD.CB17-Prkdcscid/J (Jackson Laboratory, Bar Harbor, Me.). Female micewere 5-6 weeks of age and approximately 20 grams at the time of thestudy. The HBV vector used, pWTD, is a head-to-tail dimer of thecomplete HBV genome. For a 20-gram mouse, a total injection of 1.6 mlcontaining pWTD in saline, was injected into the tail vein within 5seconds. A total of 0.3 μg of the HBV vector was injected per mouse. Inorder to allow recovery of the liver from the disruption caused by HDI,dosing of the formulated siNA compositions were started 6 days post-HDI.Encapsulated active or negative control siRNA were administered at 3mg/kg/day for three days via standard IV injection. Animals weresacrificed at 10 days following the last dose, and the levels of serumHBV DNA was measured. HBV DNA titers were determined by quantitativereal-time PCR and expressed as mean log 10 copies/ml (±SEM). Significantreductions in serum HBV DNA (FIG. 37) were observed at the 10-day timepoint in the active formulated siNA composition treated groups ascompared to both the PBS and negative control groups.

Oligonucleotide Synthesis and Characterization

All RNAs were synthesized as described herein. Complementary strandswere annealed in PBS, desalted and lyophilized. The sequences of theactive site 263 HBV siNAs are shown in below and are referenced to Sirnacompound numbers shown in FIG. 37. The modified siNAs used in vivo aretermed according to their LNP formulation, either L-086 or L-061 (seeTable IV and U.S. Provisional patent application No. 60/737,024, filedNov. 15, 2005).

The siNA sequences for active HBV siNAs are:

sense strand: (SEQ ID NO: 60) 5′ B GGAcuucucucAAuuuucuTT B 3′Compound No. 33214 antisense strand: (SEQ ID NO: 61) 5′ AGAAAAuuGAGAGAAGuccUU 3′ Compound No. 38749 antisense strand:(SEQ ID NO: 62) 5′ AGA AAAuuGAGAGAAGuccAC 3′ Compound No. 47675antisense strand: (SEQ ID NO: 63) 5′ AGA AAAuuGAGAGAAGuccTT 3′Compound No. 37793 antisense strand: (SEQ ID NO: 64) 5′ AGAAAAuuGAGAGAAGuccTsT 3′ Compound No. 35092

The siNA sequences for HBV inverted control are:

sense strand: (SEQ ID NO: 65) 5′ B ucuuuuAAcucucuucAGGTT B 3′Compound No. 33578 antisense strand: (SEQ ID NO: 66) 5′ ccuGAAGAGAGuuAAAAGATsT 3′ Compound No. 46419

(where lower case=2′-deoxy-2′-flouro; Upper Case italic=2′-deoxy; UpperCase underline=2′-O-methyl; Upper Case Bold=ribonucleotide; T=thymidine;B=inverted deoxyabasic; and s=phosphorothioate)

HBV DNA Analysis

Viral DNA was extracted from 50 mouse serum using QIAmp 96 DNA Blood kit(Qiagen, Valencia, Calif.), according to manufacture's instructions. HBVDNA levels were analyzed using an ABI Prism 7000 sequence detector(Applied Biosystems, Foster City, Calif.). Quantitative real time PCRwas carried out using the following primer and probe sequences: forwardprimer 5′-CCTGTATTCCCATCCCATCGT (SEQ ID NO: 69, HBV nucleotide2006-2026), reverse primer 5′-TGAGCCAAGAGAAACGGACTG (SEQ ID NO: 70, HBVnucleotide 2063-2083) and probe FAM 5′-TTCGCA AAATACCTATGGGAGTGGGCC (SEQID NO: 71, HBV nucleotide 2035-2062). The psHBV-1 vector, containing thefull length HBV genome, was used as a standard curve to calculate HBVcopies per mL of serum.

Example 13: Activity of LNP Formulated HBV siNA in a Mouse Model of HBVInfection

Development of therapeutic siRNA (siNA) via systemic routes ofadministration relies on both chemical modification of RNA to improvephysical stability and formulations to promote adequate tissue targetingand cell uptake. In this example, chemically modified siNA targetinghuman hepatitis B virus (HBV) was encapsulated into a liver-trophiclipid based nanoparticle and demonstrated a 2.5-3.0 log 10 reduction ofcirculating HBV DNA in mice replicating HBV. In addition, viral RNAlevels in liver were reduced by >90% as a consequence of RISC-mediatedtarget cleavage as determined by RACE analysis. This demonstrates thatchemical modification of the anti-HBV siNA is important for non-cytokinemediated knockdown of viral RNA even with nanoparticle mediateddelivery. The nanoparticle formulation delivers 65% of the siNA dose tothe liver and siNA is detectable in the liver 14 days after a singledose. Administration of these formulated siNAs to mice by intravenousinjection is well tolerated as measured by clinical chemistries,including AST and ALT levels. These results support siNA-basedtherapeutic development against important human viral pathogens of theliver such as HBV and HCV.

As described in the examples above, a number of active siNA target sitesin the HBV genome were identified in cell culture studies, with aparticularly potent siNA starting at 5′ nucleotide 263 (HBV263M) in theS-region of the HBV RNA. The HBV263 siNA molecule is described inExample 12 above and has a sense strand consisting of SEQ ID NO: 60 andan antisense strand consisting of SEQ ID NO: 64.

sense strand: (SEQ ID NO: 60) 5′ B GGAcuucucucAAuuuucuTT B 3′Compound No. 33214 antisense strand: (SEQ ID NO: 64) 5′ AGAAAAuuGAGAGAAGuccTsT 3′ Compound No. 35092

The work described in this study provides for the use of a novel lipidnanoparticle (LNP) siNA delivery technology that results in increaseddelivery of siNAs to the liver, and dramatically improves siNA potencyand duration of anti-HBV activity in vivo, including a significantreduction of HBV RNA in the liver. Furthermore, the viral RNA reductionis shown to be a direct consequence of siNA-mediated target cleavage.

Formulation of siNA

The LNP formulation utilized in the study is LNP-086 (see Table IV). ThesiNAs were incorporated in the lipid nanoparticles with highencapsulation efficiency by mixing siNA in buffer into alcoholicsolution of the lipid mixture, followed by stepwise diafiltrationprocess. The encapsulation efficiency was determined by orthogonalmethods using HPLC (Anion exchange and size exclusion chromatography)and RiboGreen assays (measuring change in fluorescence with and withoutdetergent). The particle size and charge density measurements wereperformed using a Brookhaven (Holtsville, N.Y.) ZetaPal particle sizes.

HBsAg ELISA Assay

Levels of HBsAg were determined using the Genetic Systems/Bio-Rad(Richmond, Va.) HBsAg ELISA kit, as per the manufacturer's instructions.The absorbance of cells not transfected with the HBV vector was used asbackground for the assay, and thus subtracted from the experimentalsample values.

HBV Vector-Based Mouse Model

To assess the activity of chemically stabilized siNAs against HBV,systemic dosing of the siNA was done following hydrodynamic injection(HDI) of the HBV vector in mouse strain NOD.CB17-Prkdc^(scid) (JacksonLabs, Bar Harbor, Me.). Female mice were 5-6 weeks of age andapproximately 20 g at the time of the study. The HBV vector used, pWTD,is a head-to-tail dimer of the complete HBV genome. For a 20-gram mouse,a total injection of 1.6 ml containing pWTD in saline, was injected intothe tail vein within 5 seconds. A total of 0.3 μg of the HBV vector wasinjected per mouse. Standard systemic dosing of siNAs was at 0.3 to 10mg/kg/day. In order to allow recovery of the liver from the disruptioncaused by HDI, systemic dosing was started 6 days post-HDI.

HBV DNA Analysis

Viral DNA was extracted from 50 μL mouse serum using QIAmp 96 DNA Bloodkit (Qiagen, Valencia, Calif.), according to manufacture's instructions.HBV DNA levels were analyzed using an ABI Prism 7000 sequence detector(Applied Biosystems, Foster City, Calif.). Quantitative real time PCRwas carried out using the following primer and probe sequences: forwardprimer 5′-CCTGTATTCCCATCCCATCGT (SEQ ID NO: 69, HBV nucleotide2006-2026), reverse primer 5′-TGAGCCAAGAGAAACGGACTG (SEQ ID NO: 70, HBVnucleotide 2063-2083) and probe FAM 5′-TTCGCA AAATACCTATGGGAGTGGGCC (SEQID NO: 71, HBV nucleotide 2035-2062). The psHBV-1 vector, containing thefull length HBV genome, was used as a standard curve to calculate HBVcopies per mL of serum.

HBV RNA Analysis

Total cellular RNA was isolated from approximately 100 mg mouse livertissue using Tri-Reagent (Sigma, St. Louis Mo.) according tomanufacture's instruction. HBV RNA levels were quantitated andnormalized to mouse GAPDH RNA using real time reverse-transcription(RT)-PCR in a multiplex reaction. Relative amounts of both HBV and GAPDHRNA were calculated from a standard curve of total liver RNA from an HBVinjected mouse (3-fold serial dilutions from 300 to 1 ng RNA perreaction). HBV primers and probe are described above. Mouse GAPDHprimers and probe sequences are as follows: forward primer5′-GCATCTTGGGCTACAC TGAGG (SEQ ID NO: 72, mGAPDH nucleotides 855-875),reverse primer 5′-GAAGGTGGAAGAGTGGGAGTTG (SEQ ID NO: 73, mGAPDHnucleotides 903-925), and probe VIC 5′-ACCAGGTTGTCTCCTGCGACTTCAACAG (SEQID NO: 74, mGAPDH nucleotides 876-913). Liver HBV RNA levels areexpressed as a ratio of HBV to GAPDH RNA.

5′ RACE Assay of Target RNA Cleavage

The RACE analysis was done according to the GeneRacer Kit (Invitrogen,Carlsbad, Calif.) protocol, except without prior treatment of total RNA.The total liver RNA (5 m) from animals treated with active and controlsiNA was ligated to the GeneRacer adaptor molecule. The ligated RNA wasreverse transcribed using an HBV specific primer (VSP1:5′-TGAGCCAAGAGAAACGGACTG, SEQ ID NO: 75). This was followed by PCRamplification using primers complementary to the adaptor(GR5′-5′-CGACTGGAGCACGAGGACACTGA, SEQ ID NO: 76) and HBV (VSP2:5′-GCATGGTCCCGTACTGGTTGT, SEQ ID NO: 77). The size of cleaved product(145 bp) was further confirmed by nested PCR using primers (GR5′nested5′-GGACACTGACATGGACTGAAGGAGTA, SEQ ID NO: 78) and (VSP3:5′CAGACACATCCAGCGATAACCAG, SEQ ID NO: 79) and electrophoresis on nativePAGE. The amplified product of ˜145 by was gel purified, cloned andsequenced to reveal site of siNA cleavage.

Analysis of Immune Stimulation

Five to six week old male CD-1 mice (Charles River, Wilmington, Mass.)were injected with a single 3 mg/kg dose of HBV263M-LNP or PBS controlby standard intravenous injection in the lateral tail vein. The animalswere euthanized by CO₂ inhalation followed immediately by exsanguinationat 2.5 and 8 hours after dosing (n=5 per time point). Blood wascollected through the vena cava and processed as serum for analysis. Allcytokines were quantified using sandwich ELISA kits according tomanufacturer's instructions. These were mouse IL-6, TNF-alpha, IFN-gammaand IFN-alpha (all from R&D Systems, Minneapolis, Minn.).

Pharmacokinetics

Male CD-1 mice were obtained from Charles River (Wilmington, Mass.) andweighed approximately 30 g at the time of the study. HBV263M-LNP wasadministered as a standard IV bolus (100-120 μL) at a dose ofapproximately 3 mg/kg into a lateral tail vein. Animals were euthanizedat selected timepoints (2 and 15 min; 1, 3 and 6 hours; and 1, 5, 10 and14 days after dosing) by CO₂ inhalation followed immediately byexsanguination. Blood was collected via cardiac puncture and collectedin Microtainer® brand tubes containing EDTA and plasma collected. Afterexsanguination, animals were perfused with sterile veterinary gradesaline via the heart. The liver was weighed and a sample (˜100 mg)placed in a pre-weighed homogenization tube and frozen on dry ice.

Quantitation of siNA in plasma and liver samples was done using asandwich hybridization assay with a working concentration range of0.026-6.815 ng/mL for the passenger and 0.027-6.945 ng/mL for the guidestrands. Liver samples were prepared at a concentration of 100 mg/mL intissue homogenization buffer (3 M guanidine isothiocyanate, 0.5 M NaCl,0.1 M Tris pH 7.5, 10 mM EDTA). This mixture was homogenized once inBio-101 Homogenizer (Savant, Carlsbad, Calif.) with a speed setting of6.0 and a run time of 10 sec. The homogenized liver solutions werediluted to 10 mg/ml in 1 M GITC Buffer (1 M guanidine isothiocyanate,0.5 M NaCl, 0.1 M Tris pH 7.5, 10 mM EDTA), then used in the assay atfurther dilution (1:2 to 1:10). The plasma samples were diluted >25-foldin 1 M GITC buffer. Total siNA concentrations were calculated by addingpassenger and guide strand concentrations. WinNonLin Professional (ver3.3) was used to conduct noncompartmental pharmacokinetic analysis ofresulting concentration time data.

Toxicity Evaluation

Twenty CD-1 male mice were administered the HBV263M-LNP by a single IVbolus injection at a dose of 3 mg/kg (n=10) or PBS (n=10). Body weightswere measured prior to study and prior to sample collection 1 or 14 daysafter dosing. At the appropriate timepoints, mice were euthanized by CO₂inhalation followed immediately by exsanguination (n=5/timepoint) andblood was collected for serum chemistry analysis. In addition, liver andspleen weights were collected and organ to body weight ratioscalculated.

Results

LNP Formulated HBV siNA

The LNP-086 formulation (see Table IV) was used to encapsulate activeHBV263M siNA with a sense strand consisting of SEQ ID NO: 60 and anantisense strand consisting of SEQ ID NO: 64 and a correspondinginverted control formulation of HBV263invM with a sense strandconsisting of SEQ ID NO: 65 and an antisense strand consisting of SEQ IDNO: 66.

sense strand: (SEQ ID NO: 65) 5′ B ucuuuuAAcucucuucAGGTT B 3′Compound No. 33578 antisense strand: (SEQ ID NO: 66) 5′ ccuGAAGAGAGuuAAAAGATsT 3′ Compound No. 46419

A process was developed to incorporate siNAs into the lipidnanoparticles with high efficiency by simultaneous mixing of lipid andsiNA solutions, followed by stepwise diafiltration. Using this process,the HBV263M and control HBV263Minv siNAs were encapsulated into theLNP-086 formulation. The mean siNA encapsulation efficiency was found tobe 84±2%, as determined by HPLC and RiboGreen assays. The mean particlesize was 167±10 nm, with polydispersity of 0.15±0.05. The LNP had aslight positive surface charge density of 30±2 mV.

The chemically modified HBV263M siNA encapsulated with the LNPformulation was initially assessed for activity in an HBV cell culturesystem. A single treatment of Hep G2 cells replicating HBV withHBV263M-LNP resulted in dose dependent reduction in HBsAg levels, withan IC50 of 1 nM (data not shown).

In Vivo Activity of LNP Encapsulated HBV263M

To evaluate the in vivo activity of LNP-encapsulated siNA, a mouse modelof HBV replication was used in which hydrodynamic injection (HDI) of areplication competent HBV vector results in viral replication withinhepatocytes. In this model, HBV replicates in the liver ofimmunocompromised mice for up to 80 days, resulting in detectable levelsof HBV RNA and antigens in the liver, as well as titers of HBV DNA andantigens in the serum that are similar to levels found in chronicallyinfected patients.

To assess the in vivo potency and specificity of HBV263M-LNP-086, itsactivity was compared to the control siNA HBV263invM-LNP-086.HBV-replicating mice were treated with doses of 0.3, 1, or 3 mg/kg/dayfor three days, and the levels of serum HBV DNA and HBsAg weredetermined 3 days following the last dose. A dose dependent reduction inboth HBV DNA and HBsAg serum titers was observed. Decreases in HBV DNA(FIG. 38A) serum titers of 3.0, 2.3, and 1.1 log 10 (p<0.0001) andreductions in serum HBsAg (FIG. 38B) levels of 2.4, 2.2, and 1.5 log 10(p<0.0001) were observed in the 3, 1, and 0.3 mg/kg treatment groupsrespectively, compared to the control siNA or PBS groups. Levels ofserum HBV DNA or HBsAg were equivalent in the control siNA and PBStreated groups, demonstrating the sequence specificity of the anti-HBVactivity, and the absence of non-specific lipid effects.

The duration of siNA-mediated reductions in HBV levels was examined inthe mouse model. HBV-replicating mice were treated with HBV263M-LNP-086or HBV263Minv-LNP-086 at doses of 3 mg/kg/day for three days, followedby analysis of HBV serum titers at days 3, 7, and 14 after the lastdose. The anti-HBV activity was persistent, with significant activitystill observed at day 7 (2.0 log 10 reduction) and day 14 (1.5 log 10reduction (FIG. 39). This extended persistence of siNA activity againstHBV suggested that infrequent administration of the compound could beeffective. The HBV mouse model was used to evaluate the effect of weeklydosing. Mice were treated with HBV263M-LNP-086 or HBV263Minv-LNP-086 at3 mg/kg/day on days 1 and 4 in the first week, and then once weekly foran additional three weeks. Serum HBV DNA titers were determined for days7, 14, 21, and 28. The HBV263M-LNP-086 treated groups had reductions inHBV serum titers compared to PBS treated groups of 1.7, 1.7, 1.8, and1.3 log 10 on days 7, 14, 21, and 28 respectively (FIG. 40). Theseresults suggest that the reductions in HBV titers can be maintained withweekly dosing of HBV263M-LNP-086.

Specific siNA-Mediated Cleavage of Liver HBV RNA

To examine liver specific HBV RNA cleavage mediated by the activeHBV263M-LNP-086 formulation, mice replicating HBV were treated withdoses of HBV263M-LNP-086 at 0.3, 1, 3, 10 mg/kg/day or theHBV263invM-LNP control at 10 mg/kg for three days, and levels of liverHBV RNA were determined 3 days following the last dose. Dose-dependentreduction of liver HBV RNA was observed (FIG. 41), with decreases of90%, 66.5%, 18%, and 4% seen in the 10, 3, 1, and 0.3 mg/kg HBV263M-LNPtreatment groups respectively compared to the HBV263invM-LNP-086 controlat 10 mg/kg.

To directly demonstrate that the reduction in liver HBV RNA observed inthe mouse model was due to RNAi-mediated cleavage of HBV RNA, 5′ rapidamplification of cDNA ends (RACE) analysis was used to detect cleavageof the HBV RNA at the predicted site. HBV-replicating mice were treatedwith HBV26, 3M-LNP-086 or HBV263Minv-LNP-086 at a dose of 3 mg/kg/d for3 days. The animals were sacrificed at 3, 7, or 14 days following thelast dose, and total liver RNA was isolated. Ligation of an adaptorsequence to the free 5′ends of the RNA population, and subsequent RT-PCRwith adaptor and HBV specific primers was expected to result in a PCRproduct of 145 by if the HBV RNA had been cleaved at the predictedtarget site. As shown in FIG. 42, the expected amplification product wasobserved in the HBV263 active siNA-treated samples at each time point,but not in the HBV263 control samples. PCR products were then subclonedand sequenced, confirming the correct junction between the adaptorsequence and the predicted cleavage site of the HBV263 siNA. This resultestablishes that the reduction in HBV RNA observed in the liver was dueto specific RNAi-mediated cleavage of the HBV RNA in the liver. Inaddition, the detection of specific HBV RNA cleavage products at the 7and 14 day time points demonstrates that the duration of the siNAactivity against HBV is due to continued cleavage of HBV RNA.

Analysis of siNA Induced Immunostimulation

Unmodified synthetic siNAs formulated for in vivo delivery have beenshown to induce synthesis of inflammatory cytokines and interferons in asequence specific manner, both in vitro in human peripheral bloodmononuclear cells (PBMC) and in vivo in mice. The potential for thechemically modified HBV263M-LNP-086 siNA to elicit this type of immuneresponse in comparison to an unmodified version (HBV263R-LNP-086) wasinvestigated.

sense strand: (SEQ ID NO: 67) 5′ B GGACUUCUCUCAAUUUUCUTT B 3′Compound No. 34526 antisense strand: (SEQ ID NO: 68) 5′AGAAAAUUGAGAGAAGUCCTT 3′ Compound No. 34527

CD-1 mice were injected with a single 3 mg/kg dose of HBV263M-LNP-086 orHBV263R-LNP-086. The animals were sacrificed at 2.5 or 8 hours afterdosing and blood was collected. To detect peak blood levels, IL-6 andTNF-α were measured at the 2.5 hr time point, while IFN-γ and IFN-αlevels were analyzed at 8 hrs post injection. In the HBV263M-LNP-086treated group, the mean IL-6 level was 33±21 pg/ml, a level notsignificantly different from the PBS control group at 13±4 (Table VII).In addition, in the HBV263M-LNP-086 treated group no induction of TNF-αIFN-α or IFN-γ was observed. In contrast, a significant induction of allfour cytokines was observed in the HBV263R-LNP-086 treated animals(Table VI). These results show that modified HBV263M-LNP-086 siNA didnot induce cytokines in mice, compared to the very strong responseelicited by unmodified HBV263R-LNP-086 siNA. The absence of cytokineinduction by HBV263M-LNP-086 further indicates that the anti-HBVactivity observed in the mouse model is due to specific siNA-mediatedsilencing of HBV gene expression.

Pharmacokinetics of LNP Formulated siNA

The pharmacokinetic properties of HBV263M-LNP-086 were determined inmice after a single 3 mg/kg dose. A hybridization method was used todetect the HBV263M siNA in plasma and liver over time (FIG. 43). HBV263Mwas eliminated rapidly in plasma with an elimination T112 ofapproximately 1.7 h. However, HBV263M was detected in the liverthroughout the 14 d sampling period and had an elimination T112 of 4days. A maximum concentration of 31.3±17.8 ng/mg (mean±standarddeviation) was observed in the liver at 1 hour and corresponded to65±32% of the siNA dose. At 14 days, 1.4±0.7% of the dose remainedintact in the liver. The prolonged siNA-mediated anti-HBV activityobserved in the mouse model correlates well with this extended residencetime of the siNA in the liver.

Evaluation of HBV263M-LNP Toxicity

A single dose study was conducted to determine the potential toxiceffects of HBV263M-LNP-086. Administration of HBV263M-LNP-086 was welltolerated by the animals with no morbidity or mortality. No changes inbody weight or organ to body weight ratio for liver and spleen wereobserved 1 or 14 days after administration of 3 mg/kg HBV263M-LNP (TableVII). No gross morphological changes were observed in the liver orspleen. In addition, no changes were observed in serum chemistries whichcould be attributed to administration of HBV263M-LNP-086 (Table VIII).Overall, the LNP-086 encapsulated HBV263 siNA is well tolerated at thedose level used to show significant reduction of viral titers in the HBVmouse model.

In this study, the use of a novel lipid formulation for siNA delivery isdescribed, significant improvement in delivery of siNA to the liver isdemonstrated, resulting in increased potency and long lasting reductionsin HBV titers in a mouse model of HBV infection. An excellentcorrelation was observed between the pharmacokinetic characteristics ofLNP formulated siNA, and the potency and duration of in vivo siNAactivity. Three doses at 3 mg/kg/day of HBV263M-LNP-086 reduced serumHBV DNA 2.5 to 3.0 log 10 relative to control siNA. Treatment withHBV263M-LNP-086 resulted in a significant duration of anti-HBV activitywith a 2.0 log 10 reduction in serum HBV DNA at observed at Day 7 and a1.3 log 10 reduction at Day 14.

This study also demonstrates that the use of chemically modified siRNAsencapsulated in the LNP formulation abrogates siRNA mediated inductionof cytokines in vivo. Taken together, the favorable pharmacokinetic andpotency profile of HBV263M-LNP-086 siRNA have created a potentiallytherapeutically relevant antiviral compound. This formulation deliverssiRNA effectively to the liver, and can be utilized for knockdown ofendogenous disease-associated liver targets.

Example 14: Indications

Particular conditions and disease states that can be associated withgene expression modulation include, but are not limited to cancer,proliferative, inflammatory, autoimmune, neurologic, ocular,respiratory, metabolic, dermatological, auditory, liver, kidney,infectious etc. diseases, conditions, or disorders as described hereinor otherwise known in the art, and any other diseases, conditions ordisorders that are related to or will respond to the levels of a target(e.g., target protein or target polynucleotide) in a cell or tissue,alone or in combination with other therapies.

Example 15: Multifunctional siNA Inhibition of Target RNA ExpressionMultifunctional siNA Design

Once target sites have been identified for multifunctional siNAconstructs, each strand of the siNA is designed with a complementaryregion of length, for example, of about 18 to about 28 nucleotides, thatis complementary to a different target nucleic acid sequence. Eachcomplementary region is designed with an adjacent flanking region ofabout 4 to about 22 nucleotides that is not complementary to the targetsequence, but which comprises complementarity to the complementaryregion of the other sequence (see for example FIG. 16). Hairpinconstructs can likewise be designed (see for example FIG. 17).Identification of complementary, palindrome or repeat sequences that areshared between the different target nucleic acid sequences can be usedto shorten the overall length of the multifunctional siNA constructs(see for example FIGS. 18 and 19).

In a non-limiting example, three additional categories of additionalmultifunctional siNA designs are presented that allow a single siNAmolecule to silence multiple targets. The first method utilizes linkersto join siNAs (or multifunctional siNAs) in a direct manner. This canallow the most potent siNAs to be joined without creating a long,continuous stretch of RNA that has potential to trigger an interferonresponse. The second method is a dendrimeric extension of theoverlapping or the linked multifunctional design; or alternatively theorganization of siNA in a supramolecular format. The third method useshelix lengths greater than 30 base pairs. Processing of these siNAs byDicer will reveal new, active 5′ antisense ends. Therefore, the longsiNAs can target the sites defined by the original 5′ ends and thosedefined by the new ends that are created by Dicer processing. When usedin combination with traditional multifunctional siNAs (where the senseand antisense strands each define a target) the approach can be used forexample to target 4 or more sites.

I. Tethered Bifunctional siNAs

The basic idea is a novel approach to the design of multifunctionalsiNAs in which two antisense siNA strands are annealed to a single sensestrand. The sense strand oligonucleotide contains a linker (e.g.,non-nucleotide linker as described herein) and two segments that annealto the antisense siNA strands (see FIG. 22). The linkers can alsooptionally comprise nucleotide-based linkers. Several potentialadvantages and variations to this approach include, but are not limitedto:

-   1. The two antisense siNAs are independent. Therefore, the choice of    target sites is not constrained by a requirement for sequence    conservation between two sites. Any two highly active siNAs can be    combined to form a multifunctional siNA.-   2. When used in combination with target sites having homology, siNAs    that target a sequence present in two genes (e.g., different    isoforms), the design can be used to target more than two sites. A    single multifunctional siNA can be for example, used to target RNA    of two different target RNAs.-   3. Multifunctional siNAs that use both the sense and antisense    strands to target a gene can also be incorporated into a tethered    multifuctional design. This leaves open the possibility of targeting    6 or more sites with a single complex.-   4. It can be possible to anneal more than two antisense strand siNAs    to a single tethered sense strand.-   5. The design avoids long continuous stretches of dsRNA. Therefore,    it is less likely to initiate an interferon response.-   6. The linker (or modifications attached to it, such as conjugates    described herein) can improve the pharmacokinetic properties of the    complex or improve its incorporation into liposomes. Modifications    introduced to the linker should not impact siNA activity to the same    extent that they would if directly attached to the siNA (see for    example FIGS. 27 and 28).-   7. The sense strand can extend beyond the annealed antisense strands    to provide additional sites for the attachment of conjugates.-   8. The polarity of the complex can be switched such that both of the    antisense 3′ ends are adjacent to the linker and the 5′ ends are    distal to the linker or combination thereof.    Dendrimer and Supramolecular siNAs

In the dendrimer siNA approach, the synthesis of siNA is initiated byfirst synthesizing the dendrimer template followed by attaching variousfunctional siNAs. Various constructs are depicted in FIG. 23. The numberof functional siNAs that can be attached is only limited by thedimensions of the dendrimer used.

Supramolecular Approach to Multifunctional siNA

The supramolecular format simplifies the challenges of dendrimersynthesis. In this format, the siNA strands are synthesized by standardRNA chemistry, followed by annealing of various complementary strands.The individual strand synthesis contains an antisense sense sequence ofone siNA at the 5′-end followed by a nucleic acid or synthetic linker,such as hexaethyleneglyol, which in turn is followed by sense strand ofanother siNA in 5′ to 3′ direction. Thus, the synthesis of siNA strandscan be carried out in a standard 3′ to 5′ direction. Representativeexamples of trifunctional and tetrafunctional siNAs are depicted in FIG.24. Based on a similar principle, higher functionality siNA constructscan be designed as long as efficient annealing of various strands isachieved.

Dicer Enabled Multifunctional siNA

Using bioinformatic analysis of multiple targets, stretches of identicalsequences shared between differing target sequences can be identifiedranging from about two to about fourteen nucleotides in length. Theseidentical regions can be designed into extended siNA helixes (e.g., >30base pairs) such that the processing by Dicer reveals a secondaryfunctional 5′-antisense site (see for example FIG. 25). For example,when the first 17 nucleotides of a siNA antisense strand (e.g., 21nucleotide strands in a duplex with 3′-TT overhangs) are complementaryto a target RNA, robust silencing was observed at 25 nM. 80% silencingwas observed with only 16 nucleotide complementarily in the same format.

Incorporation of this property into the designs of siNAs of about 30 to40 or more base pairs results in additional multifunctional siNAconstructs. The example in FIG. 25 illustrates how a 30 base-pair duplexcan target three distinct sequences after processing by Dicer-RNaseIII;these sequences can be on the same mRNA or separate RNAs, such as viraland host factor messages, or multiple points along a given pathway(e.g., inflammatory cascades). Furthermore, a 40 base-pair duplex cancombine a bifunctional design in tandem, to provide a single duplextargeting four target sequences. An even more extensive approach caninclude use of homologous sequences to enable five or six targetssilenced for one multifunctional duplex. The example in FIG. 25demonstrates how this can be achieved. A 30 base pair duplex is cleavedby Dicer into 22 and 8 base pair products from either end (8 b.p.fragments not shown). For ease of presentation the overhangs generatedby dicer are not shown—but can be compensated for. Three targetingsequences are shown. The required sequence identity overlapped isindicated by grey boxes. The N's of the parent 30 b.p. siNA aresuggested sites of 2′-OH positions to enable Dicer cleavage if this istested in stabilized chemistries. Note that processing of a 30mer duplexby Dicer RNase III does not give a precise 22+8 cleavage, but ratherproduces a series of closely related products (with 22+8 being theprimary site). Therefore, processing by Dicer will yield a series ofactive siNAs. Another non-limiting example is shown in FIG. 26. A 40base pair duplex is cleaved by Dicer into 20 base pair products fromeither end. For ease of presentation the overhangs generated by dicerare not shown but can be compensated for. Four targeting sequences areshown in four colors, blue, light-blue and red and orange. The requiredsequence identity overlapped is indicated by grey boxes. This designformat can be extended to larger RNAs. If chemically stabilized siNAsare bound by Dicer, then strategically located ribonucleotide linkagescan enable designer cleavage products that permit our more extensiverepertoire of multifunctional designs. For example cleavage products notlimited to the Dicer standard of approximately 22-nucleotides can allowmultifunctional siNA constructs with a target sequence identity overlapranging from, for example, about 3 to about 15 nucleotides.

Example 14: Diagnostic Uses

The siNA molecules of the invention can be used in a variety ofdiagnostic applications, such as in the identification of moleculartargets (e.g., RNA) in a variety of applications, for example, inclinical, industrial, environmental, agricultural and/or researchsettings. Such diagnostic use of siNA molecules involves utilizingreconstituted RNAi systems, for example, using cellular lysates orpartially purified cellular lysates. siNA molecules of this inventioncan be used as diagnostic tools to examine genetic drift and mutationswithin diseased cells or to detect the presence of endogenous orexogenous, for example viral, RNA in a cell. The close relationshipbetween siNA activity and the structure of the target RNA allows thedetection of mutations in any region of the molecule, which alters thebase-pairing and three-dimensional structure of the target RNA. By usingmultiple siNA molecules described in this invention, one can mapnucleotide changes, which are important to RNA structure and function invitro, as well as in cells and tissues. Cleavage of target RNAs withsiNA molecules can be used to inhibit gene expression and define therole of specified gene products in the progression of disease orinfection. In this manner, other genetic targets can be defined asimportant mediators of the disease. These experiments will lead tobetter treatment of the disease progression by affording the possibilityof combination therapies (e.g., multiple siNA molecules targeted todifferent genes, siNA molecules coupled with known small moleculeinhibitors, or intermittent treatment with combinations siNA moleculesand/or other chemical or biological molecules). Other in vitro uses ofsiNA molecules of this invention are well known in the art, and includedetection of the presence of mRNAs associated with a disease, infection,or related condition. Such RNA is detected by determining the presenceof a cleavage product after treatment with a siNA using standardmethodologies, for example, fluorescence resonance emission transfer(FRET).

In a specific example, siNA molecules that cleave only wild-type ormutant forms of the target RNA are used for the assay. The first siNAmolecules (i.e., those that cleave only wild-type forms of target RNA)are used to identify wild-type RNA present in the sample and the secondsiNA molecules (i.e., those that cleave only mutant forms of target RNA)are used to identify mutant RNA in the sample. As reaction controls,synthetic substrates of both wild-type and mutant RNA are cleaved byboth siNA molecules to demonstrate the relative siNA efficiencies in thereactions and the absence of cleavage of the “non-targeted” RNA species.The cleavage products from the synthetic substrates also serve togenerate size markers for the analysis of wild-type and mutant RNAs inthe sample population. Thus, each analysis requires two siNA molecules,two substrates and one unknown sample, which is combined into sixreactions. The presence of cleavage products is determined using anRNase protection assay so that full-length and cleavage fragments ofeach RNA can be analyzed in one lane of a polyacrylamide gel. It is notabsolutely required to quantify the results to gain insight into theexpression of mutant RNAs and putative risk of the desired phenotypicchanges in target cells. The expression of mRNA whose protein product isimplicated in the development of the phenotype (i.e., disease related orinfection related) is adequate to establish risk. If probes ofcomparable specific activity are used for both transcripts, then aqualitative comparison of RNA levels is adequate and decreases the costof the initial diagnosis. Higher mutant form to wild-type ratios arecorrelated with higher risk whether RNA levels are comparedqualitatively or quantitatively.

All patents and publications mentioned in the specification areindicative of the levels of skill of those skilled in the art to whichthe invention pertains. All references cited in this disclosure areincorporated by reference to the same extent as if each reference hadbeen incorporated by reference in its entirety individually.

One skilled in the art would readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The methodsand compositions described herein as presently representative ofpreferred embodiments are exemplary and are not, intended as limitationson the scope of the invention. Changes therein and other uses will occurto those skilled in the art, which are encompassed within the spirit ofthe invention, are defined by the scope of the claims.

It will be readily apparent to one skilled in the art that varyingsubstitutions and modifications can be made to the invention disclosedherein without departing from the scope and spirit of the invention.Thus, such additional embodiments are within the scope of the presentinvention and the following claims. The present invention teaches oneskilled in the art to test various combinations' and/or substitutions ofchemical modifications described herein toward generating nucleic acidconstructs with improved activity for mediating RNAi activity. Suchimproved activity can comprise improved stability, improvedbioavailability, and/or improved activation of cellular responsesmediating RNAi. Therefore, the specific embodiments described herein arenot limiting and one skilled in the art can readily appreciate thatspecific combinations of the modifications described herein can betested without undue experimentation toward identifying siNA moleculeswith improved RNAi activity.

The invention illustratively described herein suitably can be practicedin the absence of any element or elements, limitation or limitationsthat are not specifically disclosed herein. Thus, for example, in eachinstance herein any of the terms “comprising”, “consisting essentiallyof”, and “consisting of” may be replaced with either of the other twoterms. The terms and expressions which have been employed are used asterms of description and not of limitation, and there is no intentionthat in the use of such terms and expressions of excluding anyequivalents of the features shown and described or portions thereof, butit is recognized that various modifications are possible within thescope of the invention claimed. Thus, it should be understood thatalthough the present invention has been specifically disclosed bypreferred embodiments, optional features, modification and variation ofthe concepts herein disclosed May be resorted to by those skilled in theart, and that such modifications and variations are considered to bewithin the scope of this invention as defined by the description and theappended claims.

In addition, where features or aspects of the invention are described interms of Markush groups or other grouping of alternatives, those skilledin the art will recognize that the invention is also thereby describedin terms of any individual member or subgroup of members of the Markushgroup or other group.

TABLE I Non-limiting examples of Stabilization Chemistries forchemically modified siNA constructs Chemistry pyrimidine Purine cap p =S Strand “Stab 00” Ribo Ribo TT at S/AS 3′-ends “Stab 1” Ribo Ribo — 5at 5′-end S/AS 1 at 3′-end “Stab 2” Ribo Ribo — All Usually linkages AS“Stab 3” 2′-fluoro Ribo — 4 at 5′-end Usually 4 at 3′-end S “Stab 4”2′-fluoro Ribo 5′ and — Usually 3′-ends S “Stab 5” 2′-fluoro Ribo — 1 at3′-end Usually AS “Stab 6” 2′-O-Methyl Ribo 5′ and — Usually 3′-ends S“Stab 7” 2′-fluoro 2′-deoxy 5′ and — Usually 3′-ends S “Stab 8”2′-fluoro 2′-O-Methyl — 1 at 3′-end S/AS “Stab 9” Ribo Ribo 5′ and —Usually 3′-ends S “Stab 10” Ribo Ribo — 1 at 3′-end Usually AS “Stab 11”2′-fluoro 2′-deoxy — 1 at 3′-end Usually AS “Stab 12” 2′-fluoro LNA 5′and Usually 3′-ends S “Stab 13” 2′-fluoro LNA 1 at 3′-end Usually AS“Stab 14” 2′-fluoro 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab15” 2′-deoxy 2′-deoxy 2 at 5′-end Usually 1 at 3′-end AS “Stab 16” Ribo2′-O-Methyl 5′ and Usually 3′-ends S “Stab 17” 2′-O-Methyl 2′-O-Methyl5′ and Usually 3′-ends S “Stab 18” 2′-fluoro 2′-O-Methyl 5′ and Usually3′-ends S “Stab 19” 2′-fluoro 2′-O-Methyl 3′-end S/AS “Stab 20”2′-fluoro 2′-deoxy 3′-end Usually AS “Stab 21” 2′-fluoro Ribo 3′-endUsually AS “Stab 22” Ribo Ribo 3′-end Usually AS “Stab 23” 2′-fluoro*2′-deoxy* 5′ and Usually 3′-ends S “Stab 24” 2′-fluoro* 2′-O-Methyl* — 1at 3′-end S/AS “Stab 25” 2′-fluoro* 2′-O-Methyl* — 1 at 3′-end S/AS“Stab 26” 2′-fluoro* 2′-O-Methyl* — S/AS “Stab 27” 2′-fluoro*2′-O-Methyl* 3′-end S/AS “Stab 28” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS“Stab 29” 2′-fluoro* 2′-O-Methyl* 1 at 3′-end S/AS “Stab 30” 2′-fluoro*2′-O-Methyl* S/AS “Stab 31” 2′-fluoro* 2′-O-Methyl* 3′-end S/AS “Stab32” 2′-fluoro 2′-O-Methyl S/AS “Stab 33” 2′-fluoro 2′-deoxy* 5′ and —Usually 3′-ends S “Stab 34” 2′-fluoro 2′-O-Methyl* 5′ and Usually3′-ends S “Stab 35” 2′-fluoro** 2′-O-Methyl** Usually AS “Stab 36”2′-fluoro** 2′-O-Methyl** Usually AS “Stab 3F” 2′-OCF3 Ribo — 4 at5′-end Usually 4 at 3′-end S “Stab 4F” 2′-OCF3 Ribo 5′ and — Usually3′-ends S “Stab 5F” 2′-OCF3 Ribo — 1 at 3′-end Usually AS “Stab 7F”2′-OCF3 2′-deoxy 5′ and — Usually 3′-ends S “Stab 8F” 2′-OCF32′-O-Methyl — 1 at 3′-end S/AS “Stab 11F” 2′-OCF3 2′-deoxy — 1 at 3′-endUsually AS “Stab 12F” 2′-OCF3 LNA 5′ and Usually 3′-ends S “Stab 13F”2′-OCF3 LNA 1 at 3′-end Usually AS “Stab 14F” 2′-OCF3 2′-deoxy 2 at5′-end Usually 1 at 3′-end AS “Stab 15F” 2′-OCF3 2′-deoxy 2 at 5′-endUsually 1 at 3′-end AS “Stab 18F” 2′-OCF3 2′-O-Methyl 5′ and Usually3′-ends S “Stab 19F” 2′-OCF3 2′-O-Methyl 3′-end S/AS “Stab 20F” 2′-OCF32′-deoxy 3′-end Usually AS “Stab 21F” 2′-OCF3 Ribo 3′-end Usually AS“Stab 23F” 2′-OCF3* 2′-deoxy* 5′ and Usually 3′-ends S “Stab 24F”2′-OCF3* 2′-O-Methyl* — 1 at 3′-end S/AS “Stab 25F” 2′-OCF3*2′-O-Methyl* — 1 at 3′-end S/AS “Stab 26F” 2′-OCF3* 2′-O-Methyl* — S/AS“Stab 27F” 2′-OCF3* 2′-O-Methyl* 3′-end S/AS “Stab 28F” 2′-OCF3*2′-O-Methyl* 3′-end S/AS “Stab 29F” 2′-OCF3* 2′-O-Methyl* 1 at 3′-endS/AS “Stab 30F” 2′-OCF3* 2′-O-Methyl* S/AS “Stab 31F” 2′-OCF3*2′-O-Methyl* 3′-end S/AS “Stab 32F” 2′-OCF3 2′-O-Methyl S/AS “Stab 33F”2′-OCF3 2′-deoxy* 5′ and — Usually 3′-ends S “Stab 34F” 2′-OCF32′-O-Methyl* 5′ and Usually 3′-ends S “Stab 35F” 2′-OCF3*† 2′-O-Methyl*†Usually AS “Stab 36F” 2′-OCF3*† 2′-O-Methyl*† Usually AS CAP = anyterminal cap, see for example FIG. 10. All Stab 00-34 chemistries cancomprise 3′-terminal thymidine (TT) residues All Stab 00-34 chemistriestypically comprise about 21 nucleotides, but can vary as describedherein. All Stab 00-36 chemistries can also include a singleribonucleotide in the sense or passenger strand at the 11th base pairedposition of the double stranded nucleic acid duplex as determined fromthe 5′-end of the antisense or guide strand (see FIG. 6C) S = sensestrand AS = antisense strand *Stab 23 has a single ribonucleotideadjacent to 3′-CAP *Stab 24 and Stab 28 have a single ribonucleotide at5′-terminus *Stab 25, Stab 26, and Stab 27, Stab 35 and Stab 36 havethree ribonucleotides at 5′-terminus *Stab 29, Stab 30, Stab 31, Stab33, and Stab 34 any purine at first three nucleotide positions from5′-terminus are ribonucleotides p phosphorothioate linkage †Stab 35 has2′-O-methyl U at 3′-overhangs and three ribonucleotides at 5′-terminus†Stab 36 has 2′-O-methyl overhangs that are complementary to the targetsequence (naturually occurring overhangs) and three ribonucleotides at5′-terminus

TABLE II Compound Synthesis SEQ # # Alias Sequence ID NO: 33717 50400HBV:262U21 siNA B uGGAcuucucucAAuuuuUTTB  1 33718 50401 HBV:262U21 siNAB uGGAcuucucucAAUUUUCTTB  2 33719 50402 HBV:262U21 siNAB uGGAcuUCUCUCAAuuuucTTB  3 33720 50403 HBV:262U21 siNAB UGGACUucucucAAuuuucTTB  4 33721 50404 HBV:280L21 siNA (262C)IAAAAuuGAGAGAAGuccATsT  5 33722 50405 HBV:280L21 siNA (262C)GAAAAUuGAGAGAAGuccATsT  6 33723 50406 HBV:280L21 siNA (262C)GAAAAuUGAGAGAAGuccATsT  7 33724 50407 HBV:280L21 siNA (262C)GAAAAuuGAGAGAAGUCCATsT  8 35098 52100 HBV:280L21 siNA (262C)GAAAAuuGAGAGAAGuccATsT  9 35099 25101 HBV:280L21 siNA (262C)GAAAAuuGAGAGAAGuccATsT 10 35100 52102 HBV:280L21 siNA (262C)GAAAAuuGAGAGAAGuccATsT 11 33711 50392 HBV:263U21 siNAB GGAcuucucucAAUUUUCUTT B 12 33712 50393 HBV:263U21 siNAB GGAcuuCUCUCAAuuuucuTT B 13 33713 50394 HBV:263U21 siNAB GGACUUcucucAAuuuucuTT B 14 33714 50395 HBV:281L21 siNA (263C)AGAAAAuuGAGAGAAGuccTsT 15 33715 50396 HBV:281L21 siNA (263C)AGAAAAUUGAGAGAAGuccTsT 16 33716 50397 HBV:281L21 siNA (263C)AGAAAAuuGAGAGAAGUCCTsT 17 33703 50378 HBV:1583U21 siNAB GcAcuucGcuucAccucuITT B 18 33704 50379 HBV:1583U21 siNAB GcAcuucGcuucACCUCUGTT B 19 33705 50380 HBV:1583U21 siNAB GcAcuuCGCUUCAccucuGTT B 20 33706 50381 HBV:1583U21 siNAB GCACUUcGcuucAccucuGTT B 21 33707 50382 HBV:1601L21 siNA (1583C)UAGAGGuGAAGcGAAGuGcTsT 22 33708 50383 HBV:1601L21 siNA (1583C)CAGAGGuGAAGcGAAGuGcTsT 23 33709 50384 HBV:1601L21 siNA (1583C)cAGAGGUGAAGCGAAGuGcTST 24 33710 50385 HBV:1601L21 siNA (1583C)cAGAGGuGAAGcGAAGUGCTsT 25 35075 52063 HBV:281L21 siNA (263C)AGAAAAUUGAGAGAAGUCCTT 26 35076 52064 HBV:281L21 siNA (263C)AGAAAAuuGAGAGAAGUCCTT 27 35077 52065 HBV:281L21 siNA (263C)AGAAAAUUGAGAGAAGuccTT 28 34714 51673 HBV:263U21 siNA GGACUUCUCUCAAuuuucuTT 29 34715 51674 HBV:263U21 siNA GGACUUcucucAAUUUUCUTT 30 34716 51675 HBV:263U21 siNA GGAcuuCUCUCAAUUUUCUTT 31 35086 52088 HBV:263U21 siNA B GGAcuucucucAAuuuucUTT B 32 35088 52090 HBV:263U21 siNA B GGAcuucucucAAuuuucCTT B 33 35087 52089 HBV:263U21 siNA B GGAcuucucucAAuuuuCUTT B 34 35090 52092 HBV:281L21 siNAAGAAAAuuGAGAGAAGuccTsT 35 35091 52093 HBV:281L21 siNAAGAAAAuuGAGAGAAGuccTsT 36 35092 52094 HBV:281L21 siNAAGAAAAuuGAGAGAAGuccTsT 37 35093 52095 HBV:281L21 siNAAGAAAAuuGAGAGAAGuccTsT 38 35094 52096 HBV:281L21 siNAAGAAAAuuGAGAGAAGuccTsT 39 30607 HBV:262U21 siNA B uGGAcuucucucAAuuuucTTB 40 33214 HBV:263U21 siNA B GGAcuucucucAAuuuucuTT B 41 32429 HBV:1583U21 siNA B GcAcuucGcuucAccucuGTT B 42 33591 HBV:263U21 siNA B GGACUUCUCUCAAUUUUCUTT B 43 33593 HBV:281L21 siNAAGAAAAUUGAGAGAAGUCCTsT 44 33701 HBV:263U21 siNAB UCUUUUAACUCUCUUCAGGTT B 45 33702 HBV:281L21 siNA CCUGAAGAGAGUUAAAAGATsT 46 32448 HBV:1583U21 siNA B GCACUUCGCUUCACCUCUGTTB 47 32458 HBV:1601L21 siNA CAGAGGUGAAGCGAAGUGCTsT 48 32488 HBV:1583U21 siNA B GUCUCCACUUCGCUUCACGTT B 49 32498 HBV:1601L21 siNACGUGAAGCGAAGUGGAGACTsT 50 33139 56164 HCVa:282U21B GcGAAAGGccuuGuGGuAcTT B 51 38279 56171 HCVa:300L21GUAccAcAAGGccuuucGcTsT 52 38296 56172 HCVa:300L21G uAccAcAAGGccuuucGcTsT 53 33149 56158 HCVa:304U21B cuGAuAGGGuGcuuGcGAGTT B 54 33189 56119 HCVa:322L21cucGcAAGcAcccuAucAGTsT 55 35180 52274 HCVa:322L21 CUCGcAAGcAcccuAucAGTsT56 31703 56161 HCVa:327U21 B ccGGGAGGucucGuAGAcc TT B 57 35175 56124HCVa:345L21 GGUcuAcGAGAccucccGGTsT 58 35176 56127 HCVa:345L21GGucuAcGAGAccucccGGTsT 59 33214 49777 HBV:263U21 siRNAB GGAcuucucucAAuuuucuTT B 60 38749 56694 HBV:281L21 siRNAAGAAAAuuGAGAGAAGuccUU 61 47675 62734 HBV:281L21 siRNAAGAAAAuuGAGAGAAGuccAC 62 37793 55512 HBV:281L21 siRNAAGAAAAuuGAGAGAAGuccTT 63 35092 52094 HBV:281L21 siRNAAGAAAAuuGAGAGAAGuccTsT 64 33578 50194 HBV:263U21 siRNAB ucuuuuAAcucucuucAGGTT B 65 35092 52094 HBV:281L21 siRNAAGAAAAuuGAGAGAAGuccTsT 66 34526 51436 HBV:263U21 siRNAGGACUUCUCUCAAUUUUCUTT 67 34527 51437 HBV:281L21 siRHAAGAAAAUUGAGAGAAGUCCTT 68 UPPER CASE = Ribonucleotide lower case =2′-deoxy-2′-fluoro UNDERLINE = 2′-O-methyl ITALIC = 2′-deoxy B =inverted deoxyabasic s = phosphorothioate I = Inosine

TABLE III Wait Wait Wait Reagent Equivalents Amount Time* DNA Time*2′-O-methyl Time* RNA A. 2.5 μmol Synthesis Cycle ABI 394 InstrumentPhosphoramidites 6.5 163 μL 45 sec 2.5 min 7.5 min S-Ethyl Tetrazole23.8 238 μL 45 sec 2.5 min 7.5 min Acetic Anhydride 100 233 μL 5 sec 5sec 5 sec N-Methyl Imidazole 186 233 μL 5 sec 5 sec 5 sec TCA 176 2.3 mL21 sec 21 sec 21 sec Iodine 11.2 1.7 mL 45 sec 45 sec 45 sec Beaucage12.9 645 μL 100 sec 300 sec 300 sec Acetonitrile NA 6.67 mL NA NA NA B.0.2 μmol Synthesis Cycle ABI 394 Instrument Phosphoramidites 15 31 μL 45sec 233 sec 465 sec S-Ethyl Tetrazole 38.7 31 μL 45 sec 233 min 465 secAcetic Anhydride 655 124 μL 5 sec 5 sec 5 sec N-Methyl Imidazole 1245124 μL 5 sec 5 sec 5 sec TCA 700 732 μL 10 sec 10 sec 10 sec Iodine 20.6244 μL 15 sec 15 sec 15 sec Beaucage 7.7 232 μL 100 sec 300 sec 300 secAcetonitrile NA 2.64 mL NA NA NA C. 0.2 μmol Synthesis Cycle 96 wellInstrument Equivalents: DNA/ Amount: DNA/2′-O- Wait Wait Wait Reagent2′-O-methyl/Ribo methyl/Ribo Time* DNA Time* 2′-O- methyl Time* RiboPhosphoramidites 22/33/66 40/60/120 μL 60 sec 180 sec 360 sec S-EthylTetrazole  70/105/210 40/60/120 μL 60 sec 180 min 360 sec AceticAnhydride 265/265/265 50/50/50 μL 10 sec 10 sec 10 sec N-MethylImidazole 502/502/502 50/50/50 μL 10 sec 10 sec 10 sec TCA 238/475/475250/500/500 μL 15 sec 15 sec 15 sec Iodine 6.8/6.8/6.8 80/80/80 μL 30sec 30 sec 30 sec Beaucage 34/51/51 80/120/120 100 sec 200 sec 200 secAcetonitrile NA 1150/1150/1150 μL NA NA NA Wait time does not includecontact time during delivery. Tandem synthesis utilizes double couplingof linker molecule

TABLE IV Lipid Nanoparticle (LNP) Formulations For- mula- tion #Composition Molar Ratio L051 CLinDMA/DSPC/Chol/PEG-n-DMG 48/40/10/2 L053DMOBA/DSPC/Chol/PEG-n-DMG 30/20/48/2 L054 DMOBA/DSPC/Chol/PEG-n-DMG50/20/28/2 L069 CLinDMA/DSPC/Cholesterol/PEG-Cholesterol 48/40/10/2 L073pCLinDMA or CLin DMA/DMOBA/DSPC/ 25/25/20/28/2 Chol/PEG-n-DMG L077eCLinDMA/DSPC/Cholesterol/2KPEG-Chol 48/40/10/2 L080eCLinDMA/DSPC/Cholesterol/2KPEG-DMG 48/40/10/2 L082pCLinDMA/DSPC/Cholesterol/2KPEG-DMG 48/40/10/2 L083pCLinDMA/DSPC/Cholesterol/2KPEG-Chol 48/40/10/2 L086CLinDMA/DSPC/Cholesterol/2KPEG-DMG/ 43/38/10/2/7 Linoleyl alcohol L061DMLBA/Cholesterol/2KPEG-DMG 52/45/3 L060 DMOBA/Cholesterol/2KPEG-DMG N/Pratio of 5 52/45/3 L097 DMLBA/DSPC/Cholesterol/2KPEG-DMG  50/20/28 L098DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 3 52/45/3 L099DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 4 52/45/3 L100 DMOBA/DOBA/3%PEG-DMG, N/P ratio of 3 52/45/3 L101 DMOBA/Cholesterol/2KPEG-Cholesterol52/45/3 L102 DMOBA/Cholesterol/2KPEG-Cholesterol, N/P 52/45/3 ratio of 5L103 DMLBA/Cholesterol/2KPEG-Cholesterol 52/45/3 L104CLinDMA/DSPC/Cholesterol/2KPEG-cholesterol/ 43/38/10/2/7 Linoleylalcohol L105 DMOBA/Cholesterol/2KPEG-Chol, N/P ratio of 2 52/45/3 L106DMOBA/Cholesterol/2KPEG-Chol, N/P ratio of 3 67/30/3 L107DMOBA/Cholesterol/2KPEG-Chol, N/P ratio of 52/45/3 1.5 L108DMOBA/Cholesterol/2KPEG-Chol, N/P ratio of 2 67/30/3 L109DMOBA/DSPC/Cholesterol/2KPEG-Chol, N/P 50/20/28/2 ratio of 2 L110DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 52/45/3 1.5 L111DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 67/30/3 1.5 L112DMLBA/Cholesterol/2KPEG-DMG, N/P ratio of 52/45/3 1.5 L113DMLBA/Cholesterol/2KPEG-DMG, N/P ratio of 67/30/3 1.5 L114DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 2 52/45/3 L115DMOBA/Cholesterol/2KPEG-DMG, N/P ratio of 2 67/30/3 L116DMLBA/Cholesterol/2KPEG-DMG, N/Pratio of 2 52/45/3 L117DMLBA/Cholesterol/2KPEG-DMG, N/P ratio of 2 52/45/3 N/P ratio =Nitrogen:Phosphorous ratio between cationic lipid and nucleic acid

TABLE V Table V: Sirna algorithm describing patterns with their relativescore for predicting hyperactive siNAs. All the positions given are forthe sense strand of 19-mer siNA. Description of pattern Pattern # ScoreG or C at position 1 1 5 A or U at position 19 2 10 A/U rich betweenposition 15-19 3 10 String of 4 Gs or 4 Cs (not preferred) 4 −100 G/Crich between position 1-5 5 10 A or U at position 18 6 5 A or U atposition 10 7 10 G at position 13 (not preferred) 8 −3 A at position 139 3 G at position 9 (not preferred) 10 −3 A at position 9 11 3 A or U atposition 14 12 10

TABLE VI Immunostimluation in CD-1 mice treated with a single 3 mg/kginjection of LNP formulated siRNA. IL-6 and TNF-α levels were measuredat 2.5 hrs post injection, while IFN-γ and IFN-α, levels were measuredat 8 hrs post treatment. Values are shown as mean ± standard deviation,n = 5 IL-6 TNF-α IFN-γ IFN-α siRNA (pg/mL) (pg/mL) (pg/mL) (pg/mL) PBS13 ± 4 BLOD^(a) BLOQ^(b) BLOD HBV263M-  33 ± 21 BLOD  BLOQ  BLOD LNP-086HBV263R- 2035 ± 378 169 ± 61 756 ± 345 41822 ± 11321 LNP-086^(a)BLOD—Below limit of detection ^(b)BLOQ—Below limit of quantitation

TABLE VII Body and organ weights 1 and 14 days after administration of 3mg/kg HBV263-LNP-086 or PBS in mice. Five animals per dose group wereeuthanized per timepoint. Body weight was collected just prior toeuthanasia. Values are shown as mean ± standard deviation. Days PostBody Liver:Body Spleen Weight Spleen to Body Dose Dose Weight (g) LiverWt (g) Weight (g/g) (g) Weight (g/g) PBS 1 34.5 ± 3.3^(a) 2.119 ± 0.1780.057 ± 0.002 0.125 ± 0.022 0.003 ± 0.001 14 36.5 ± 3.5  1.991 ± 0.2750.055 ± 0.002 0.110 ± 0.034 0.003 ± 0.001 HBV263- 1 33.6 ± 2.8^(a) 1.970± 0.119 0.055 ± 0.003 0.106 ± 0.017 0.003 ± 0.001 LNP-086 14 35.1 ± 1.2 1.944 ± 0.101 0.055 ± 0.001 0.112 ± 0.025 0.003 ± 0.001 3 mg/kg ^(a)n =10 for body weight 1 day after dosing

TABLE VIII Serum chemistry values 1 and 14 days after administration of3 mg/kg HBV263-LNP-086 or PBS in mice. Values are shown as mean ±standard deviation, n = 5. Days Total Post Alk Phos ALT AST AlbuminTotal Protein Globulin bilirubin BUN Cholesterol Glucose Dose Dose (U/L)(U/L) (U/L) (g/dL) (g/dL) (g/dL) (mg/dL) (mg/dL) (mg/dL) (mg/dL) PBS 1129 ± 22 39 ± 6  46 ± 5  2.9 ± 0.1 5.4 ± 0.2 2.5 ± 0.1 0.1 ± 0.0 25 ± 4185 ± 28 242 ± 14 14 170 ± 66 48 ± 12 48 ± 5  3.0 ± 0.1 5.4 ± 0.2 2.5 ±0.2 0.2 ± 0.0 28 ± 4 195 ± 20 242 ± 25 HBV263- 1 154 ± 33 44 ± 14 58 ±21 2.9 ± 0.2 5.6 ± 0.3 2.7 ± 0.1 0.2 ± 0.1 27 ± 2 167 ± 11 259 ± 30LNP-086 14 141 ± 72 40 ± 14 50 ± 10 3.0 ± 0.1 5.5 ± 0.1 2.5 ± 0.1 0.2 ±0.0 28 ± 2 178 ± 20 274 ± 48 3 mg/kg

What I claim is:
 1. A chemically modified multifunctional shortinterfering nucleic acid (siNA) assembled from two separatedouble-stranded siNAs, each double-stranded siNA having a sense strandand an antisense strand of a length from about 18 to about 28nucleotides, wherein either (a) one end of each sense strand of the twodouble-stranded siNAs is covalently bonded via a tether, or (b) one endof each antisense strand of the two double-stranded siNAs is covalentlybonded via a tether, wherein the antisense strand of eachdouble-stranded siNA comprises a sequence complementary to a targetsequence or a portion thereof, wherein the sense or antisense strand ofeach double-stranded siNA is annealed to its corresponding strand thathas been tethered at one end to another strand of the otherdouble-stranded siNA, wherein the tether is a nucleotide linker or anon-nucleotide linker, and wherein the chemically modifiedmultifunctional siNA comprises one or more chemical modifications. 2.The chemically modified multifunctional siNA of claim 1, wherein theantisense strand of each double-stranded siNA comprises a sequencecomplementary to a first and second target sequence, respectively, or aportion thereof, and wherein the first target sequence or a portionthereof is different than the second target sequence or a portionthereof.
 3. The chemically modified multifunctional siNA of claim 2,wherein the first and second target sequences are present in the sametarget nucleic acid molecule.
 4. The chemically modified multifunctionalsiNA of claim 2, wherein the first and second target sequences arepresent in different target nucleic acid molecules.
 5. The chemicallymodified multifunctional siNA of claim 1, wherein the tether covalentlybonds the 3′-ends of the sense strands of the two double-stranded siNAsor bonds the 5′-ends of the sense strands of the two double-strandedsiNAs.
 6. The chemically modified multifunctional siNA of claim 1,wherein the tether covalently bonds the 5′-end of one sense strand ofone double-stranded siNA to the 3′-end of the sense strand of the otherdouble-stranded siNA.
 7. The chemically modified multifunctional siNA ofclaim 1, wherein the tether covalently bonds the 3′-ends of theantisense strands of the two double-stranded siNAs or bonds the 5′-endsof the antisense strands of the two double-stranded siNAs.
 8. Thechemically modified multifunctional siNA of claim 1, wherein the tethercovalently bonds the 5′-end of one antisense strand of onedouble-stranded siNA to the 3′-end of the antisense strand of the otherdouble-stranded siNA.
 9. The chemically modified multifunctional siNA ofclaim 1, further comprising one or more terminal phosphate groups at theend of the antisense strand(s) of one double-stranded siNA or bothdouble-stranded siNAs.
 10. The chemically modified multifunctional siNAof claim 1, wherein the tether is a biodegradable linker.
 11. Thechemically modified multifunctional siNA of claim 10, wherein the tetheris a biodegradable nucleic acid linker molecule containing 2 to 20nucleotides in length.
 12. The chemically modified multifunctional siNAof claim 11, wherein the tether contains ribonucleotides;deoxyribonucleotides; 2′-modified nucleotides selected from the groupconsisting of 2′-O-methyl, 2′-fluoro, 2′-amino, 2′-O-amino, 2′-C-allyl,and 2′-O-allyl; or combination thereof.
 13. The chemically modifiedmultifunctional siNA of claim 1, wherein the tether contains anon-nucleotide linker selected from the group consisting of an abasicnucleotide, polyether, polyamine, polyamide, peptide, carbohydrate,lipid, polyhydrocarbon, and combinations thereof.
 14. The chemicallymodified multifunctional siNA of claim 1, wherein the sense strand(s)and antisense strand(s) of one double-stranded siNA or bothdouble-stranded siNAs have a length from about 19 to about 23nucleotides.
 15. The chemically modified multifunctional siNA of claim1, further comprising a second tether covalently bonding either one endof each sense strand of the two double-stranded siNAs or one end of eachantisense strand of the two double-stranded siNAs, so that the sensestrands of the two double-stranded siNAs have one end covalently bondedby one tether and the antisense strands of the two double-stranded siNAshave one end covalently bonded by another tether, respectively.
 16. Thechemically modified multifunctional siNA of claim 15, wherein the secondtether is a non-nucleotide linker.
 17. The chemically modifiedmultifunctional siNA of claim 16, wherein the non-nucleotide linker isselected from the group consisting of an abasic nucleotide, polyether,polyamine, polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, andcombinations thereof.
 18. The chemically modified multifunctional siNAof claim 1, wherein the one or more chemical modifications are selectedfrom the group consisting of phosphorothioate internucleotide linkage,2′-deoxy modification, 2′-O-methyl modification, 2′-deoxy-2′-fluoromodification, 4′-thio modification, 2′-O-trifluoromethyl modification,2′-O-ethyl-trifluoromethoxy modification, 2′-O-difluoromethoxy-ethoxymodification, 2′-deoxy-2′-fluoroarabino modification, universal basemodification, acyclic modification, 5-C-methyl modification, glycerylmodification, abasic modification, inverted deoxy abasic modification,and combinations thereof.
 19. A pharmaceutical composition comprisingthe chemically modified multifunctional siNA of claim 1, alone or incombination with a pharmaceutically acceptable carrier or excipient. 20.A method for inhibiting the expression of a target gene comprising thestep of administering the chemically modified multifunctional siNA ofclaim 1, in an amount sufficient to inhibit expression of the targetgene.